WO2010058860A1

WO2010058860A1 - Measurement method and measurement reagent for c-reactive protein in sample

Info

Publication number: WO2010058860A1
Application number: PCT/JP2009/069898
Authority: WO
Inventors: 穴田哲也; 中村雄樹
Original assignee: 株式会社シノテスト
Priority date: 2008-11-18
Filing date: 2009-11-18
Publication date: 2010-05-27
Also published as: JP5807300B2; JPWO2010058860A1

Abstract

Provided are a new measurement method and a measurement reagent for measuring C-reactive protein in a sample by measuring a complex aggregate formed through a specific binding reaction between the C-reactive protein and a specific binding substance that binds to the C-reactive protein and is fixed to a carrier. The method and the measurement reagent enable the measurement range to be expanded from low concentrations to high concentrations and enable the concentration of the C-reactive protein to be measured accurately over a wide range from low concentrations to high concentrations. To measure the C-reactive protein in a sample, a specific binding substance that binds to C-reactive protein dependent on calcium ions and a specific binding substance that binds to C-reactive protein independent of calcium ions are fixed to a carrier, and a complex aggregate comprising said specific binding substances and the C-reactive protein, which is formed through said specific binding reaction between said specific binding substances fixed to the carrier and the C-reactive protein contained in the sample, is measured.

Description

Method and reagent for measuring C-reactive protein in sample

The present invention relates to a method and reagent for measuring C-reactive protein in a sample.
The present invention is particularly useful in fields such as chemistry, life science, analytical chemistry, and clinical testing.

A method for measuring a small amount of test substance contained in a sample using a reaction between substances having specific affinity such as antigen and antibody, sugar and lectin, nucleotide chain and complementary nucleotide chain, and ligand and receptor. Various measuring reagents are known.
Among these, immunological measurement methods using an antigen-antibody reaction (immune reaction) between an antigen and an antibody are widely practiced.
For example, C-reactive protein (hereinafter also abbreviated as CRP) is a protein that exhibits a precipitation reaction with C polysaccharides of pneumococcal bacteria, and an increase in blood CRP concentration may cause infections. It is evidence that the patient is afflicted or that inflammation is occurring in the body.
For this reason, CRP has been used as a marker for various inflammations in order to diagnose diseases such as infections or inflammation.
As a measuring method of this CRP, a method for observing a specific reaction with C polysaccharide, and an antibody that binds to CRP (anti-CRP antibody) as a reagent, an antigen between CRP in the sample and anti-CRP antibody in the reagent Examples thereof include a method for producing an antigen-antibody complex by an antibody reaction.
At present, methods such as an immunonephelometric method, an immunoturbidimetric method, a latex immunoturbidimetric method, and the like, which are methods for generating an antigen-antibody complex with an anti-CRP antibody, are widely used in daily examinations.
In addition, this CRP is called a calcium binding protein and has a binding site with calcium ions in its three-dimensional structure, and CRP (calcium-binding CRP) bound to calcium ions has its three-dimensional structure (antigenicity). Is known to change part of [When a chelating agent such as EDTA coexists with CRP, calcium ions bound to CRP are bound to this chelating agent and become “calcium non-binding CRP”. ]
For example, when an animal is inoculated with CRP to obtain an antiserum against CRP, CRP binds to calcium ions in the blood of the animal to form calcium-binding CRP, and the animal inoculated with CRP has calcium-binding CRP. Produces antibodies against various antigenic determinants.
In addition, the produced antibody includes “an antibody that binds to calcium-binding CRP but has a significantly reduced binding activity to calcium-unbinding CRP” and “calcium-binding CRP and calcium-unbinding CRP”. It is known that there are two types of antibodies, “antibodies that bind equally to each other” (see, for example, Non-Patent Document 1).
"Clinical Pathology", Vol. 32, No. 2, pp. 223-224, published in 1984

The CRP concentration in the serum (or plasma) of healthy people is generally 0.3 mg / dL or less, but it rises by a sensitive reaction in a short time to inflammation and bacterial infection, and is 2,000 to 4,000 times Also reach.
Since CRP concentration correlates with the magnitude and extent of inflammation or its improvement, the clinical significance of the measurement is great.
Further, even if the CRP concentration is in the normal range (0.3 mg / dL or less), it is reported that the possibility of developing coronary artery disease is high when the concentration is relatively high, and has attracted attention.
For this reason, in measuring CRP in a sample, it has been required to accurately measure CRP in a wide range of concentrations from low concentration to high concentration.
In addition, in the CRP measurement in this sample, as a method for accurately measuring CRP in a wide range from a low concentration to a high concentration, for example, a method using a mixture of two or more types of latex particles having different average particle diameters ( For example, Patent Document 1), a method of adding a surfactant to a measurement reagent (for example, see Patent Document 2), a method of using a combination of a polyclonal antibody and a monoclonal antibody (for example, see Patent Document 3), etc. have been proposed. ing.
JP-A 63-65369 JP 2001-318099 A JP 2006-17745 A

Therefore, an object of the present invention is to measure CRP in a sample by measuring a complex aggregate produced by a specific binding reaction between CRP and a specific binding substance that binds to CRP immobilized on a carrier. A measuring method and a measuring reagent that can expand the measuring range from a low concentration to a high concentration and accurately measure a wide range of CRP from a low concentration to a high concentration. That is.

As a result of intensive studies aimed at solving the above-mentioned problems, the inventors of the present invention have found that complex aggregation is caused by a specific binding reaction between CRP and a specific binding substance that binds to CRP immobilized on a carrier. In a method and a measurement reagent for measuring CRP in a sample by measuring a substance, a carrier on which a specific binding substance that binds to CRP is immobilized in a calcium ion-dependent manner is used to measure CRP in the sample. When performed, when CRP is in the low concentration range, the reactivity with CRP is low and the sensitivity of the measurement is low, but the reactivity with CRP is high from the middle concentration range to the high concentration range, which increases the CRP concentration. In response to this, it was found that the complex aggregates also increased, that is, the measurement was quantitative from the medium concentration range to the high concentration range.
In addition, when CRP in a sample was measured using a carrier on which a specific binding substance that binds to CRP independent of calcium ions was used, when CRP was in a low concentration range, CRP and The reactivity of CRP is high and the sensitivity of measurement is high, but the reactivity with CRP decreases from the medium concentration range to the high concentration range, and the complex aggregate does not increase or decreases even when the CRP concentration increases. However, it was found that there was no quantitative measurement in the middle to high concentration range.
The present inventors use a combination of two specific binding substances, a specific binding substance that binds to CRP in a calcium ion-dependent manner and a specific binding substance that binds to CRP in a calcium ion-independent manner. As a result, it was found that CRP can be measured with a wide range of concentrations from low concentration to high concentration by extending the measurement range from low concentration to high concentration, and the present invention has been completed.
That is, the present invention provides the following inventions.
(1) A specific binding substance that binds to a C-reactive protein in a calcium ion-dependent manner and a specific binding substance that binds to a C-reactive protein in a calcium ion-independent manner are immobilized on a carrier, respectively, By measuring a complex aggregate of the specific binding substance and the C-reactive protein produced by the specific binding reaction between the specific binding substance and the C-reactive protein contained in the sample. A method for measuring C-reactive protein.
(2) The C-reactive protein in the sample according to (1), wherein the specific binding substance that binds to the C-reactive protein in a calcium ion-dependent manner is an antibody that binds to the C-reactive protein in a calcium-ion-dependent manner How to measure.
(3) The method for measuring C-reactive protein in a sample according to (1) or (2) above, wherein the antibody that binds to C-reactive protein in a calcium ion-dependent manner is a monoclonal antibody.
(4) Any of (1) to (3) above, wherein the specific binding substance that binds to C-reactive protein independent of calcium ions is an antibody that binds to C-reactive protein independent of calcium ions. A method for measuring C-reactive protein in a sample according to item 1.
(5) The method for measuring C-reactive protein in a sample according to any one of (1) to (4), wherein the antibody that binds to C-reactive protein independent of calcium ions is a monoclonal antibody.
(6) The method for measuring C-reactive protein in a sample according to any one of (1) to (5), wherein the carrier is latex particles.
(7) A specific binding substance that binds to C-reactive protein in a calcium ion-dependent manner and a carrier on which a specific binding substance that binds to C-reactive protein in a calcium ion-independent manner is immobilized. A reagent for measuring C-reactive protein in a sample.
(8) The C-reactive protein in the sample according to (7), wherein the specific binding substance that binds to the C-reactive protein in a calcium ion-dependent manner is an antibody that binds to the C-reactive protein in a calcium-ion-dependent manner Measuring reagent.
(9) The reagent for measuring C-reactive protein in the sample according to (7) or (8), wherein the antibody that binds to C-reactive protein in a calcium ion-dependent manner is a monoclonal antibody.
(10) Any of the above (7) to (9), wherein the specific binding substance that binds to C-reactive protein independent of calcium ions is an antibody that binds to C-reactive protein independent of calcium ions. A reagent for measuring C-reactive protein in a sample according to item 1.
(11) The reagent for measuring C-reactive protein in a sample according to any one of (7) to (10), wherein the antibody that binds to C-reactive protein independent of calcium ions is a monoclonal antibody.
(12) The reagent for measuring C-reactive protein in a sample according to any one of (7) to (11), wherein the carrier is latex particles.

According to the present invention, a method for measuring CRP in a sample by measuring a complex aggregate produced by a specific binding reaction between CRP and a specific binding substance that binds to CRP immobilized on a carrier. And a specific binding substance that binds to CRP in a calcium ion-dependent manner and a specific binding substance that binds to CRP in a calcium ion-independent manner in combination in the measurement reagent, In the measurement of CRP, the measurement range can be expanded from a low concentration to a high concentration, and CRP in a wide range from a low concentration to a high concentration can be accurately measured.

FIG. 1 shows the binding of anti-CRP antibodies to calcium-bound CRP and calcium-unbound CRP.
FIG. 2 is a diagram showing the binding of anti-CRP antibodies to calcium-bound CRP and calcium-unbound CRP.
FIG. 3 is a diagram showing the binding of anti-CRP antibodies to calcium-bound CRP and calcium-unbound CRP.
FIG. 4 shows the binding of anti-CRP antibodies to calcium-bound CRP and calcium-unbound CRP.
FIG. 5 is a graph showing the difference in absorbance obtained by measuring CRP in a sample using a measurement reagent containing latex particles to which each anti-CRP antibody is immobilized.
FIG. 6 shows latex particles immobilized with an antibody (antibody-1) that binds to CRP in a calcium ion-dependent manner, and latex particles immobilized with an antibody (antibody-4) that binds to CRP in a calcium ion-independent manner. It is the figure which showed the light absorbency difference obtained by measuring CRP in a sample using the containing measurement reagent.
FIG. 7 shows latex particles immobilized with an antibody (antibody-2) that binds to CRP in a calcium ion-dependent manner, and latex particles immobilized with an antibody (antibody-4) that binds to CRP in a calcium ion-independent manner. It is the figure which showed the light absorbency difference obtained by measuring CRP in a sample using the containing measurement reagent.
FIG. 8 shows latex particles in which an antibody that binds to CRP in a calcium ion-dependent manner (antibody-3) is immobilized, and latex particles in which an antibody that binds to CRP in a calcium ion-independent manner (antibody-4) is immobilized. It is the figure which showed the light absorbency difference obtained by measuring CRP in a sample using the containing measurement reagent.
FIG. 9 is a diagram showing the binding of anti-CRP antibodies to calcium-bound CRP and calcium-unbound CRP.
FIG. 10 is a graph showing the difference in absorbance obtained by measuring CRP in a sample using a measurement reagent containing latex particles on which an anti-CRP antibody is immobilized.
FIG. 11 shows latex particles immobilized with an antibody (antibody-1) that binds to CRP in a calcium ion-dependent manner and latex particles immobilized with an antibody (antibody-4) that binds to CRP in a calcium ion-independent manner. It is the figure which showed the light absorbency difference obtained by measuring CRP in a sample using the containing measurement reagent.
FIG. 12 shows latex particles immobilized with an antibody (antibody-2) that binds to CRP in a calcium ion-dependent manner, and latex particles immobilized with an antibody (antibody-4) that binds to CRP in a calcium ion-independent manner. It is the figure which showed the light absorbency difference obtained by measuring CRP in a sample using the containing measurement reagent.
FIG. 13 shows latex particles immobilized with an antibody (antibody-3) that binds to CRP in a calcium ion-dependent manner, and latex particles immobilized with an antibody (antibody-4) that binds to CRP in a calcium ion-independent manner. It is the figure which showed the light absorbency difference obtained by measuring CRP in a sample using the containing measurement reagent.
FIG. 14 shows latex particles immobilized with an antibody (antibody-1) that binds to CRP in a calcium ion-dependent manner and latex particles immobilized with an antibody (antibody-5) that binds to CRP in a calcium ion-independent manner. It is the figure which showed the light absorbency difference obtained by measuring CRP in a sample using the containing measurement reagent.
FIG. 15 shows latex particles immobilized with an antibody (antibody-2) that binds to CRP in a calcium ion-dependent manner, and latex particles immobilized with an antibody (antibody-5) that binds to CRP in a calcium ion-independent manner. It is the figure which showed the light absorbency difference obtained by measuring CRP in a sample using the containing measurement reagent.
FIG. 16 shows latex particles immobilized with an antibody (antibody-3) that binds to CRP in a calcium ion-dependent manner, and latex particles immobilized with an antibody (antibody-5) that binds to CRP in a calcium ion-independent manner. It is the figure which showed the light absorbency difference obtained by measuring CRP in a sample using the containing measurement reagent.

Hereinafter, the present invention will be described in detail.
[1] Specific binding substance that binds to C-reactive protein
In the present invention, the specific binding substance that binds to the C-reactive protein is a substance that can specifically bind to CRP, and is particularly limited as long as it is a substance that can specifically bind to CRP. There is no.
Examples of the specific binding substance that binds to CRP include an antibody that can bind to CRP (anti-CRP antibody), an aptamer (nucleic acid aptamer or peptide aptamer), an affibody, or a receptor.
Examples of antibodies that can bind to CRP (anti-CRP antibodies) include, for example, monoclonal antibodies, polyclonal antibodies, antisera, and antibody fragments [Fab and F (ab ′)) that can bind to CRP. ₂ Etc.], or single chain antibodies (scFv) and the like.
The specific binding substance that binds to CRP is preferably an antibody that can bind to CRP (anti-CRP antibody), and more preferably the antibody is a monoclonal antibody.
[2] Specific binding substance that binds to C-reactive protein in a calcium ion-dependent manner
In the present invention, a specific binding substance that binds to C-reactive protein in a calcium ion-dependent manner (hereinafter sometimes abbreviated as “Ca-dependent CRP-specific binding substance”) binds to calcium-binding CRP. Non-binding CRP refers to a specific binding substance whose binding activity is significantly reduced.
For example, as described above, in the presence of calcium ions, CRP bound to calcium ions changes a part of its three-dimensional structure. When a chelating agent such as EDTA is present in the sample, the calcium ion bound to CRP binds to this chelating agent, and the three-dimensional structure (antigenicity) of CRP changes. End up.
In this case, the binding activity of the specific binding substance that binds to CRP in a calcium ion-dependent manner is significantly lower than that of CRP (calcium non-binding CRP) whose steric structure has been changed by the presence of a chelating agent.
From this, the specific binding substance that binds to CRP in a calcium ion-dependent manner is a part in which the three-dimensional structure is changed by binding of CRP to calcium ion (if the specific binding substance is an antibody, this changing part is It is presumed to be a specific binding substance that recognizes antigenic determinants.
The specific binding substance that binds to C-reactive protein in a calcium ion-dependent manner is not particularly limited as long as it is a substance that can specifically bind to CRP in a calcium ion-dependent manner.
Examples of the substance capable of specifically binding to CRP in a calcium ion-dependent manner include, for example, an antibody, aptamer (nucleic acid aptamer or peptide aptamer), affibody, or receptor capable of binding to CRP in a calcium ion-dependent manner. Etc.
Examples of antibodies that can bind to CRP in a calcium ion-dependent manner include, for example, monoclonal antibodies, polyclonal antibodies, antisera, and antibody fragments [Fab or F (ab ') ₂ Etc.], or single chain antibodies (scFv) and the like.
In addition, the antibody capable of binding to CRP in a calcium ion-dependent manner is an antibody (chimeric antibody, human that has been changed to an amino acid sequence of an animal species different from the animal immunizing the immunogen (CRP) by genetic recombination technology or the like. Or a fully humanized antibody).
The specific binding substance that binds to CRP in a calcium ion-dependent manner is preferably an antibody that can bind to CRP in a calcium ion-dependent manner, and more preferably the antibody is a monoclonal antibody.
In the present invention, two or more kinds of specific binding substances that bind to CRP in a calcium ion-dependent manner may be used.
And the specific binding substance couple | bonded with CRP dependently on calcium ion can be suitably prepared by a well-known method etc.
[3] Specific binding substance that binds to C-reactive protein independent of calcium ion
In the present invention, a specific binding substance that binds to a C-reactive protein in a calcium ion-independent manner (hereinafter sometimes abbreviated as “Ca-independent CRP-specific binding substance”) is either a calcium-binding CRP or a non-calcium-binding CRP. Bound CRP refers to a specific binding substance that binds to approximately the same extent.
That is, for example, as described above, in the presence of calcium ions, CRP bound to calcium ions changes a part of its three-dimensional structure. When a chelating agent such as EDTA is present in the sample, the calcium ion bound to CRP binds to this chelating agent, and the three-dimensional structure (antigenicity) of CRP changes. End up.
In this case, the specific binding substance that binds to CRP in a calcium ion-independent manner is CRP in which the three-dimensional structure has been changed by the presence of a chelating agent (non-calcium-binding CRP) and CRP to which calcium ions are bound. (Calcium-binding CRP) can bind to almost the same extent.
A specific binding substance that binds to CRP in a calcium ion-independent manner is a part other than the part where the steric structure is changed by binding of CRP to calcium ion (if the specific binding substance is an antibody, this part is determined by antigen It is speculated that this is a specific binding substance that recognizes the group).
The specific binding substance that binds to CRP independently of calcium ions is not particularly limited as long as it is a substance that can specifically bind to CRP independent of calcium ions.
Examples of substances that can specifically bind to CRP independent of calcium ions include, for example, antibodies, aptamers (nucleic acid aptamers or peptide aptamers), affibodies that can bind to CRP independent of calcium ions, Or a receptor etc. can be mentioned.
Examples of antibodies that can bind to CRP independent of calcium ions include, for example, monoclonal antibodies, polyclonal antibodies, antisera, and antibody fragments [Fab or F that can bind to CRP independent of calcium ions. (Ab ') ₂ Etc.], or single chain antibodies (scFv) and the like.
Further, this antibody capable of binding to CRP independent of calcium ions is an antibody (chimeric antibody, which has been changed to an amino acid sequence of an animal species different from an animal immunizing an immunogen (CRP) by genetic recombination technology or the like. It may be a humanized antibody or a fully humanized antibody).
The specific binding substance that binds to CRP independent of calcium ions is preferably an antibody that can bind to CRP independent of calcium ions, and more preferably the antibody is a monoclonal antibody.
In the present invention, two or more kinds of specific binding substances that bind to CRP independently of calcium ions may be used.
And the specific binding substance couple | bonded with CRP independent of calcium ion can be suitably prepared by a well-known method etc.
[4] A method for preparing a polyclonal antibody that binds to CRP in a calcium ion-dependent manner or a polyclonal antibody that binds to CRP in a calcium ion-independent manner
A polyclonal antibody that binds to CRP in a calcium ion-dependent manner or a polyclonal antibody that binds to CRP in a calcium ion-independent manner can be prepared by the following procedure.
As an immunogen for antibody production, all or part of CRP such as natural CRP, CRP obtained by genetic recombination, or a CRP-derived peptide can be used.
The above-mentioned immunogen, or the conjugate of the above-mentioned immunogen and carrier is used for mammals (mouse, nude mouse, rabbit, rat, sheep, goat, cow, horse, camel, etc.) or birds (chicken, ostrich, etc.) Immunize.
The amount of immunization of the immunogen or the conjugate of the immunogen and the carrier is determined by the type of immunized animal, the site of immunization, etc. Each mouse is injected with 0.1 μg to 5 mg, preferably 50 μg to 2 mg of the immunogen or a conjugate of the immunogen and a carrier.
In the case of rabbits, 10 μg to 500 mg of the immunogen or a combination of the immunogen and a carrier is immunized once per rabbit.
The immunogen or the combined immunogen and carrier is preferably added and mixed with an adjuvant for immunization injection. As the adjuvant, known ones such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant or pertussis adjuvant can be used.
Immunization may be performed at a site such as subcutaneous, intravenous, intraperitoneal or back.
After the initial immunization, booster injections of the immunogen or a conjugate of the immunogen and the carrier are given at sites such as subcutaneous, intravenous, intraperitoneal or back at intervals of 2 to 3 weeks. Also in this case, the immunogen or the conjugate of the immunogen and the carrier is preferably boosted by adding an adjuvant and mixing.
After the first immunization, the antibody titer in the serum of the immunized animal is repeatedly measured by ELISA or the like. When the antibody titer reaches a plateau, whole blood is collected, and the serum is separated to obtain an antiserum containing the antibody.
The antiserum is subjected to antibody purification by a salting-out method using ammonium sulfate, sodium sulfate or the like, ion exchange chromatography, gel filtration method or affinity chromatography, or a combination of these methods to obtain a polyclonal antibody.
The polyclonal antibody obtained here includes both a polyclonal antibody that binds to CRP in a calcium ion-dependent manner and a polyclonal antibody that binds to CRP in a calcium ion-independent manner. Is passed through an affinity chromatography column immobilized as a ligand on a solid phase in the presence of a chelating agent.
Polyclonal antibodies that bind to CRP in a calcium ion-independent manner bind to the solid phase via the ligand (non-calcium-bound CRP) of this column and are collected.
In contrast, a polyclonal antibody that binds to CRP in a calcium ion-dependent manner passes through this column without binding to the ligand (non-calcium-binding CRP) of this column. A polyclonal antibody that binds to CRP in a calcium ion-dependent manner can be obtained.
In addition, after a polyclonal antibody that binds to CRP in a calcium ion-dependent manner passes through, a polyclonal antibody that binds to the CRP in a calcium ion-independent manner bound to a ligand (non-calcium-bound CRP) of this column is treated with a low pH solution. Alternatively, a polyclonal antibody that binds to CRP in a calcium ion-independent manner can be obtained by releasing it from the ligand by a conventional method such as passing a solution containing a salt through a column and obtaining this fraction.
In addition, when animals are immunized using a conjugate of an immunogen and a carrier, antibodies against this carrier are present in the obtained polyclonal antibody. Therefore, removal of the antibody against such a carrier should be performed. Is preferred.
As this removal treatment method, a carrier is added to the obtained polyclonal antibody solution to remove aggregates generated, or the carrier is immobilized on an insolubilized solid phase and removed by affinity chromatography. Can do.
[5] Preparation of monoclonal antibody that binds to CRP in a calcium ion-dependent manner, or a monoclonal antibody that binds to CRP in a calcium ion-independent manner
A monoclonal antibody that binds to CRP in a calcium ion-dependent manner or a monoclonal antibody that binds to CRP in a calcium ion-independent manner can be prepared by the following procedure.
Monoclonal antibodies can be produced using a hybridoma produced by Keller et al.'S cell fusion method (G. Koehler et al., Nature, 256, 495-497, published in 1975) using Epstein-Barr virus (EB virus) that causes lymphoma. It can be obtained by a method for increasing lymphocytes, a method for genetically manipulating microorganisms such as cultured cells derived from mouse bone marrow or yeast.
Preparation of a monoclonal antibody by the cell fusion method can be performed by the following operation.
As an immunogen for antibody production, all or part of CRP such as natural CRP, CRP obtained by genetic recombination, or a CRP-derived peptide can be used.
The immunogen, or the conjugate of the immunogen and the carrier is used in a mammal (mouse, nude mouse, rabbit, rat, sheep, goat, cow, horse, camel, etc., for example, BALB / c of an inbred mouse. ) Or birds (such as chickens or ostriches).
The immunization amount of the immunogen or the conjugate of the immunogen and the carrier can be appropriately determined depending on the type of immunized animal, the site of immunization, and the like. Preferably, 0.1 μg to 5 mg, preferably 50 μg to 2 mg of the immunogen or a combination of the immunogen and a carrier is immunized at a time.
The immunogen or the conjugate of the immunogen and the carrier is preferably immunized by adding an adjuvant and mixing.
Known adjuvants such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide adjuvant, or pertussis adjuvant can be used as the adjuvant.
Immunization may be performed at a site such as subcutaneous, intravenous, intraperitoneal or back.
After the first immunization, booster injections of the immunogen or a conjugate of the immunogen and the carrier are given at sites of subcutaneous, intravenous, intraperitoneal, or back at 1-2 week intervals.
The number of booster injections is generally 2 to 6 times.
Also in this case, the immunogen or the combined immunogen and carrier is preferably boosted by adding an adjuvant and mixing.
After the first immunization, the antibody titer in the serum of the immunized animal is repeatedly measured by ELISA (enzyme immunoassay) or the like. When the antibody titer reaches a plateau, the immunogen or the immunogen and the carrier A solution obtained by dissolving the conjugate in physiological saline (0.9% sodium chloride aqueous solution) is injected intravenously or intraperitoneally to obtain the final immunization. Three to five days after the final immunization, cells having antibody-producing ability such as spleen cells, lymph node cells or peripheral lymphocytes of immunized animals are obtained.
The cell having antibody-producing ability obtained from this immunized animal is fused with myeloma cells (myeloma cells) of mammals (mouse, nude mouse, rat, etc.). A cell line deficient in an enzyme such as guanine phosphoribosyl transferase (HGPRT) or thymidine kinase (TK) is preferred. For example, the P3-X63-Ag8 strain (ATCC) which is a HGPRT-deficient cell line derived from BALB / c mice. TIB9), P3-X63-Ag8-U1 strain (Cancer Research Source Bank (JCRB) 9085), P3-NS1-1-Ag4-1 strain (JCRB 0009), P3-X63-Ag8.653 strain (JCRB 0028) Alternatively, SP2 / O-Ag-14 strain (JCRB 0029) or the like is used. be able to.
Cell fusion can be performed using a fusion promoter such as polyethylene glycol (PEG) of various molecular weights, liposomes or Sendai virus (HVJ), or by electrofusion.
When myeloma cells are of HGPRT deficient strain or TK deficient strain, fusion of cells having antibody-producing ability and myeloma cells by using a selection medium (HAT medium) containing hypoxanthine, aminopterin, and thymidine Only cells (hybridomas) can be selectively cultured and propagated.
The hybridoma culture supernatant thus obtained was measured for its binding to calcium-bound CRP and calcium-unbound CRP by ELISA as described later, and a monoclonal antibody produced from the hybridoma was obtained. Production of a monoclonal antibody that binds to CRP in a calcium ion-dependent manner by determining whether it is a monoclonal antibody that binds to CRP in a calcium ion-dependent manner or a monoclonal antibody that binds to CRP in a calcium ion-independent manner. And a hybridoma producing a monoclonal antibody that binds to CRP in a calcium ion-independent manner.
By combining this hybridoma selection method with a known cloning method such as limiting dilution, a monoclonal antibody-producing cell line that binds to CRP in a calcium ion-dependent manner or a monoclonal antibody that binds to CRP in a calcium ion-independent manner Each antibody-producing cell line can be isolated and obtained.
By culturing this monoclonal antibody-producing cell line in an appropriate medium, a monoclonal antibody that binds to CRP in a calcium ion-dependent manner or a monoclonal antibody that binds to CRP in a calcium ion-independent manner can be obtained from the culture supernatant. However, a serum-free medium or a low-concentration serum medium may be used as the medium. In this case, a medium such as DMEM medium, RPMI 1640 medium, or ASF medium 103 is preferably used because the antibody can be easily purified. it can.
In addition, a monoclonal antibody-producing cell line can be injected into the abdominal cavity of a mammal that is compatible with this and previously stimulated with pristane or the like, and the monoclonal antibody can be obtained from ascites collected in the abdominal cavity after a certain period of time. .
The monoclonal antibody thus obtained was purified by a salting-out method using ammonium sulfate, sodium sulfate or the like, a method such as ion exchange chromatography, gel filtration or affinity chromatography, or a combination of these methods. It can be obtained as a monoclonal antibody.
[6] Determination of a specific binding substance that binds to CRP in a calcium ion-dependent manner or a specific binding substance that binds to CRP in a calcium ion-independent manner
(1) About judgment
Whether the specific binding substance that binds to CRP is a specific binding substance that binds to CRP in a calcium ion-dependent manner or a specific binding substance that binds to CRP in a calcium ion-independent manner depends on the CRP. Determination is performed by measuring the binding property of the specific binding substance to be bound to calcium-bound CRP and the binding property to non-calcium-bound CRP.
A “specific binding substance that binds to CRP”, which binds to calcium-bound CRP but has a markedly reduced binding activity to non-calcium-bound CRP, is a specific binding substance that binds to CRP in a calcium ion-dependent manner. .
In addition, a “specific binding substance that binds to CRP” that binds to approximately the same degree in both calcium-bound CRP and non-calcium-bound CRP is a specific binding substance that binds to CRP independent of calcium ions.
(2) Specific example of determination
Hereinafter, this determination will be described with a more specific example.
(B) Preparation of reagents
(A) Preparation of CRP-immobilized microplate
CRP is brought into contact with each well of a 96-well microtiter plate, adsorbed and immobilized to prepare a CRP-immobilized microplate.
(B) Preparation of enzyme-labeled antibody
An “enzyme-labeled antibody” is prepared by labeling an enzyme with an antibody that can specifically bind to a “specific binding substance that binds to CRP” to be determined. [For example, when the specific binding substance that binds to CRP is an anti-CRP mouse antibody, an enzyme-labeled anti-mouse IgG antibody or the like can be used as this enzyme-labeled antibody. ]
(C) Preparation of cleaning solution
A surfactant is dissolved in pure water to prepare a cleaning solution.
(D) Preparation of color developing solution
By using the enzyme-labeled antibody as a labeling enzyme catalyst, a color developing solution containing a substance related to a reaction that causes color development is prepared.
(B) Sample
(A) Preparation of diluent A
A calcium salt is dissolved in pure water to prepare a calcium ion-containing aqueous solution.
(B) Preparation of diluent B
A chelating agent is dissolved in pure water to prepare a chelating agent-containing aqueous solution.
(C) Sample of specific binding substance containing calcium ions and binding to CRP
The solution containing the “specific binding substance that binds to CRP” to be determined is diluted with the diluent A in (a) above, and a “sample of specific binding substance that binds to CRP and contains calcium ions” is prepared. Prepare.
The diluent A in (a) is a sample that does not contain any “specific binding substance that binds to CRP”, that is, a negative sample.
(D) Sample of specific binding substance containing chelating agent and binding to CRP
The solution containing the “specific binding substance that binds to CRP” to be determined is diluted with the diluent B in (b) above, and a “sample of a specific binding substance that contains chelating agent and binds to CRP” is prepared. Prepare.
The diluent B in (b) is a sample that does not contain any “specific binding substance that binds to CRP”, that is, a negative sample.
(C) Measurement
(A) The “sample of a specific binding substance that binds to CRP containing calcium ions” of (c) of (b) is added to the well of the CRP-immobilized microplate of (a) of (a). .
As a result, the CRP immobilized in this well becomes a calcium-binding CRP bonded to calcium ions.
Then, it is allowed to stand for a certain period of time, and a specific binding reaction between the specific binding substance that binds to CRP contained in the sample and the calcium binding type CRP immobilized in the well is performed.
(B) The “sample of a specific binding substance that binds to a chelating agent-containing CRP” in (d) of (b) above is placed in the other well of the CRP-immobilized microplate of (a) in (a). Added.
As a result, the CRP immobilized in this well becomes a calcium non-binding CRP.
Then, it is allowed to stand for a certain period of time, and a specific binding reaction between the specific binding substance that binds to CRP contained in the sample and the calcium non-binding CRP immobilized on the well is performed.
(C) After the liquid in each well is sucked and removed, the washing liquid of (c) (c) is added to each well, and this is removed by suction and washed. This washing operation is repeated several times.
(D) Add the enzyme-labeled antibody of (b) to (b) to each of the wells and leave it for a certain time.
As a result, the enzyme-labeled antibody is bound to the “specific binding substance that binds to CRP” bound to the well of the CRP-immobilized microplate via calcium-bound CRP or calcium-unbound CRP.
(E) After sucking and removing the liquid in each well, the washing liquid is added to each well, and this is sucked and removed to perform washing. This washing operation is repeated several times.
(F) Next, the color developing solution (d) of (b) is added to each well and allowed to stand for a certain period of time.
Thus, the “enzyme-labeled antibody” labeled enzyme bound to the well of the CRP-immobilized microplate via “specific binding substance that binds to CRP” and “calcium-bound CRP or calcium-unbound CRP” Then, a color development reaction is performed by a reaction with a substance contained in the color development solution.
(G) The absorbance of the liquid in each well is measured using a microplate reader or the like at a wavelength suitable for measuring the color development.
(H) Except that the negative sample is used in place of the sample, the procedure is performed as in (a) to (g), and the absorbance is measured.
(I) The absorbance difference value calculated by subtracting the absorbance measured in (h) from the absorbance measured in (g) in the well in which calcium-binding CRP is immobilized is obtained.
In this measurement system, the absorbance difference value reflects the binding property of the “specific binding substance that binds to CRP” with “calcium-binding CRP”. The higher the absorbance difference value is, It shows that the binding property of “specific binding substance that binds to CRP” with “calcium-binding CRP” is high.
The lower this absorbance difference value is, the lower the binding property of “specific binding substance that binds to CRP” with “calcium-binding CRP” is.
(J) Moreover, the value of the absorbance difference calculated by subtracting the absorbance measured in (h) from the absorbance measured in (g) in the well in which the calcium non-binding CRP is immobilized is obtained.
In this measurement system, the value of the absorbance difference is a value reflecting the binding of the “specific binding substance that binds to CRP” with “calcium non-binding CRP”, and the higher this absorbance difference value, It shows that the “specific binding substance that binds to CRP” has high binding property to “calcium non-binding CRP”.
The lower the absorbance difference value, the lower the binding property of “specific binding substance that binds to CRP” with “calcium non-binding CRP”.
(K) Determination of whether a specific binding substance that binds to CRP is a specific binding substance that binds to CRP in a calcium ion-dependent manner or a specific binding substance that binds to CRP in a calcium ion-independent manner. As described above, “specific binding substance that binds to CRP”, which binds to calcium-bound CRP but has a significantly reduced binding activity to non-calcium-bound CRP, is expressed as “calcium ion-dependent CRP. It is determined that it is a “specific binding substance that binds”.
In addition, regarding “specific binding substance that binds to CRP” that binds to approximately the same extent in both calcium-binding CRP and non-calcium binding CRP, “specific binding substance that binds to CRP independent of calcium ions” It is determined that
(L) In the measurement operations (a) to (h), the well in which the calcium-binding CRP used for measurement is fixed and the well in which the calcium non-binding CRP is fixed are the same microtiter. It is preferably a plate well.
The measurement operations (a) to (h) are preferably performed continuously without taking any time.
Specifically, this determination can also be performed as follows.
(3) Judgment by measuring multiple samples
(A) When making a determination in accordance with the description of “specific example of determination” in (2) above, measuring a plurality of samples, that is, a plurality of samples with different concentrations of specific binding substances that bind to CRP Measurement is preferably performed.
(B) This specific example is as described in [Example 1] described later. Samples of “specific binding substances that bind to CRP” at a plurality of concentrations are used in the above-mentioned “specific examples of determination”. The value of the difference in absorbance obtained by measurement according to the description of ”is plotted on the vertical axis, and the concentration of“ specific binding substance that binds to CRP ”of the measured sample is plotted on the horizontal axis, and calcium-binding CRP is immobilized. A graph (figure) in which the measured value (absorbance difference) in the well and the measured value (absorbance difference) in the well in which the calcium non-binding CRP is immobilized is plotted is prepared.
(C) In this graph, a curve connecting measured values (absorbance difference) in wells in which “calcium-binding CRP” is immobilized, that is, “calcium-binding CRP” of “specific binding substance that binds to CRP” in the sample. ”And a curve connecting the measurement value (absorbance difference) in the well in which“ calcium non-binding CRP ”is immobilized, that is,“ specific binding substance that binds to CRP ”in the sample. Comparison is made with a curve showing the binding property with “non-calcium-bound CRP”.
(D) Here, in this graph, the above-mentioned “curve showing the binding property with calcium-binding CRP” and the above-mentioned “curve showing the binding property with calcium-non-binding CRP” are largely separated from each other. The “specific binding substance that binds to CRP” in the sample is determined to be “specific binding substance that binds to CRP in a calcium ion-dependent manner”.
(E) In the graph, is the above-mentioned “curve showing the binding property with calcium-binding CRP” and the above “curve showing the binding property with calcium-non-binding CRP” not separated from each other? Alternatively, if they are slightly separated from each other, the “specific binding substance that binds to CRP” in the sample is determined to be “specific binding substance that binds to CRP independent of calcium ions”.
(4) Further judgment method
When determination is difficult as described above, the specific binding substance that binds to CRP is “a specific binding substance that binds to CRP in a calcium ion-dependent manner” or “independent of calcium ions” as follows. It is possible to determine whether or not it is a “specific binding substance that binds to CRP”.
(A) First, according to the description of “determination by measuring a plurality of samples” in the above (3), a curve indicating the binding property of the “specific binding substance that binds to CRP” in the sample with “calcium-binding CRP” "Curve connecting measured values [absorbance difference] in wells immobilized with calcium-bound CRP" and "Curve showing binding property with calcium-unbound CRP" (well with immobilized calcium-unbound CRP) A graph (figure) showing the measured value [absorbance difference] is shown.
(B) Next, when the “curve showing the binding property with calcium-binding CRP” in this graph increases as the concentration of “specific binding substance that binds to CRP” in the sample increases and reaches a plateau or maximum The value of the difference in absorbance (A) is obtained.
(C) Next, in the “curve showing the binding property with calcium-binding CRP” in this graph, the value of the absorbance difference when the value of the absorbance difference reaches the plateau or maximum (1/2) of the absorbance difference value (A). Value (C1) of the “specific binding substance that binds to CRP” is obtained.
(D) In addition, in the “curve showing the binding property with calcium non-binding CRP” in this graph, the “difference in absorbance” is the value of “A / 2” in (c) above. The value (C2) of the concentration of “specific binding substance” is determined.
(E) Next, the value of “C1” is compared with the value of “C2” to obtain the value of “C2 / C1”.
(F) When the value of “C2 / C1” is 3 or more, the “specific binding substance that binds to CRP” in the sample is “specific binding substance that binds to CRP in a calcium ion-dependent manner”. It is determined that
Further, when the value of “C2 / C1” is less than 3, “specific binding substance that binds to CRP” in the sample is “specific binding substance that binds to CRP independent of calcium ions”. Is determined.
(G) In addition, in the “curve showing the binding property to calcium non-binding CRP” in this graph, the concentration of “specific binding substance that binds to CRP” in the sample on the horizontal axis is obtained in (c) above. In the range up to the value of “3 * C1” obtained by multiplying the value of “C1” by three times, the “curve showing the binding property to the calcium non-binding CRP” is the value of “A / 2” in (c). Is not equal to the value of “C2 / C1” being equal to or greater than 3, the “specific binding substance that binds to CRP” in the sample is expressed as “calcium ion dependent CRP. It is determined that it is a “specific binding substance that binds”.
[7] A specific binding substance that binds to CRP in a calcium ion-dependent manner and a carrier on which a specific binding substance that binds to CRP in a calcium ion-independent manner is immobilized
In the present invention, the specific binding substance that binds to CRP in a calcium ion-dependent manner and the carrier on which the specific binding substance that binds to CRP in a calcium ion-independent manner is bound to CRP in the calcium ion-dependent manner. And a specific binding substance that binds to CRP independent of calcium ions is immobilized on a carrier.
The specific binding substance that binds to CRP in a calcium ion-dependent manner and the carrier on which the specific binding substance that binds to CRP in a calcium ion-independent manner is immobilized has a specific binding that binds to CRP in a calcium ion-dependent manner. It may be a mixture of a carrier on which a substance is immobilized and a carrier on which a specific binding substance that binds to CRP independent of calcium ions is immobilized.
In this case, the mixing ratio of the carrier immobilizing a specific binding substance that binds to CRP in a calcium ion-dependent manner and the carrier immobilizing a specific binding substance that binds to CRP in a calcium ion-independent manner depends on the mixing ratio used. The specific binding substance and the type of carrier to be used vary depending on the conditions and the like, and thus cannot be generally stated. For example, it is possible to use a mixture in which both carriers have the same concentration.
Further, a specific binding substance that binds to CRP in a calcium ion-dependent manner and a carrier on which a specific binding substance that binds to CRP in a calcium ion-independent manner is immobilized has a specific binding that binds to CRP in a calcium ion-dependent manner. The substance and a specific binding substance that binds to CRP independent of calcium ions may be immobilized on the same carrier.
In this case, the ratio between the specific binding substance that binds to CRP in a calcium ion-dependent manner and the specific binding substance that binds to CRP in a calcium ion-independent manner depends on the specific binding substance used and the type of carrier used. Since it differs depending on the conditions, etc., it cannot be generally stated. For example, it can be mentioned that both specific binding substances are immobilized on a carrier so as to have the same ratio.
A specific binding substance that binds to CRP in a calcium ion-dependent manner and a carrier on which a specific binding substance that binds to CRP in a calcium ion-independent manner is immobilized has a specific specificity that binds to CRP in a calcium ion-dependent manner. A carrier having a binding substance immobilized thereon, a carrier having a specific binding substance which binds to CRP independent of calcium ions, a specific binding substance which binds to CRP in a calcium ion dependent manner, and calcium The specific binding substance that binds to CRP in an ion-independent manner may be a mixture of those immobilized on the same carrier.
The material of this carrier is not particularly limited. For example, polystyrene, styrene-styrene sulfonate copolymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylic acid ester copolymer, vinyl acetate-acrylic acid copolymer. Polymer, polyacrolein, styrene-methacrylic acid copolymer, styrene-glycidyl (meth) acrylic acid copolymer, styrene-butadiene copolymer, methacrylic acid polymer, acrylic acid polymer, gelatin, silica, alumina, carbon Examples thereof include black, metal compounds, metals, ceramics, and magnetic materials.
The carrier may be particles such as latex particles, liposomes, microcapsules, or red blood cells.
The carrier in the present invention is preferably particles, and particularly preferably latex particles.
Immobilization of a specific binding substance that binds to CRP in a calcium ion-dependent manner and a specific binding substance that binds to CRP in a calcium ion-independent manner is carried out by physical adsorption, chemical binding, or these It can carry out by well-known methods, such as combined use.
In the case of the physical adsorption method, according to a known method, a specific binding substance that binds to CRP in a calcium ion-dependent manner and / or a specific binding substance that binds to CRP in a calcium ion-independent manner and a carrier are buffered. A specific binding substance that binds to CRP in a calcium ion-dependent manner and / or a specific binding substance that binds to CRP in a calcium ion-independent manner dissolved in a buffer solution, etc. It can be carried out by contacting with a carrier.
When the chemical binding method is used, the “Clinical Pathology Special Issue 53: Immunoassay for Clinical Examinations—Technology and Applications” edited by the Japanese Society of Clinical Pathology, Clinical Pathology Publications, 1983; Japan Biochemical Society In accordance with a known method described in ed. “Shinsei Kagaku Kenkyu Ken 1 Protein IV”, published by Tokyo Kagaku Dojin, published in 1991, etc. A specific binding substance that binds to CRP in a calcium ion-dependent manner, and a carrier that is mixed with and contacted with a bivalent cross-linking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide And / or a specific binding substance that binds to CRP in a calcium ion-independent manner; Hexyl group, a thiol group, can be carried out such as by reacting a bifunctional crosslinking reagent of the aldehyde group or a hydroxyl group.
A specific binding substance that binds to CRP in a calcium ion-dependent manner, and a carrier on which a specific binding substance that binds to CRP in a calcium ion-independent manner is bound, binds to CRP in a calcium ion-dependent manner. In the case of a mixture of a carrier immobilizing a substance and a carrier immobilizing a specific binding substance that binds to CRP in a calcium ion-independent manner, it binds to CRP in a calcium ion-dependent manner by the method described above. Examples thereof include a method in which a specific binding substance is immobilized on a carrier, a specific binding substance that binds to CRP independent of calcium ions is immobilized on the carrier, and these two kinds of carriers are mixed.
In addition, a specific binding substance that binds to CRP in a calcium ion-dependent manner and a carrier on which a specific binding substance that binds to CRP in a calcium ion-independent manner is immobilized binds to CRP in a calcium ion-dependent manner. When the substance and the specific binding substance that binds to CRP independent of calcium ions are immobilized on the same carrier, the specific binding substance that binds to CRP dependently of calcium ions, and A specific binding substance that binds to CRP in a calcium ion-dependent manner by preparing a solution containing both of the specific binding substances that bind to CRP in a calcium ion-independent manner and bringing the solution into contact with a carrier by the above-described method or the like And a method of immobilizing a specific binding substance that binds to CRP in a calcium ion-independent manner on the same carrier.
Furthermore, in order to suppress the specific binding substance that binds to CRP in a calcium ion-dependent manner and the carrier on which the specific binding substance that binds to CRP in a calcium ion-independent manner is immobilized, non-specific reaction, etc. If necessary, the bovine serum albumin is immobilized on the surface of a carrier on which a specific binding substance that binds to CRP in a calcium ion-dependent manner and a specific binding substance that binds to CRP in a calcium ion-independent manner is immobilized. (BSA), casein, gelatin, egg white albumin or a salt thereof, a known method such as contact with a protein, a surfactant, skim milk powder or the like to coat, and a carrier blocking treatment (masking treatment) is performed. May be.
In addition, when measuring CRP in a sample in the present invention by a turbidimetric method such as a latex turbidimetric method, the size (particle size) of a carrier such as latex particles is not particularly limited.
However, a specific binding substance that binds to CRP in a calcium ion-dependent manner and a carrier on which a specific binding substance that binds to CRP in a calcium ion-independent manner is immobilized is a complex with CRP contained in the sample. Because of the degree of generation of aggregates (aggregates) and the ease of measurement of the generated composite aggregates, the size (particle diameter) of the carrier is 0 (average particle diameter). The thickness is preferably 0.01 μm to 10 μm, more preferably 0.04 μm to 1 μm.
In the CRP measurement method and measurement reagent in the sample of the present invention, two or more types of carriers different in size (particle size), material, shape, etc. may be used as the carrier.
In addition, when measuring CRP in a sample according to the present invention by a turbidimetric method such as latex turbidimetric method, a specific binding substance that binds to CRP in a calcium ion-dependent manner, and a CRP independent of calcium ions. The concentration of the carrier on which the specific binding substance that binds to the carrier is immobilized during the measurement reaction is the distribution density of the specific binding substance on the carrier surface, the size of the carrier (particle size), and the mixture of the sample and the measurement reagent. Since the optimum concentration differs depending on various conditions such as the ratio, it cannot be generally stated.
However, usually, a sample and a measurement reagent are mixed, a specific binding substance that binds to CRP in a calcium ion-dependent manner immobilized on a carrier, and a specific binding substance that binds to CRP in a calcium ion-independent manner; A specific binding substance that binds to CRP in a calcium ion-dependent manner and a specific binding substance that binds to CRP in a calcium ion-independent manner during a measurement reaction in which a specific binding reaction with CRP contained in the sample is performed In general, the concentration of the carrier on which the carrier is immobilized is 0.005 to 1% (w / v) in the reaction mixture during the measurement reaction. A specific binding substance that binds to CRP in a calcium ion-dependent manner and a specific binding substance that binds to CRP in a calcium ion-independent manner are fixed. It is preferable to include a standardized carrier in the measurement reagent.
In the measuring method and the measuring reagent of the present invention, a specific binding substance that binds to CRP in a calcium ion-dependent manner and a carrier on which a specific binding substance that binds to CRP in a calcium ion-independent manner is immobilized is used as bovine serum albumin (BSA), proteins such as human serum albumin (HSA), casein or salts thereof; various metal ions such as calcium ions; various salts such as calcium salts; various saccharides; skim milk powder; various animal sera such as normal rabbit serum; Various preservatives such as sodium phosphide or antibiotics; activating substances; reaction promoting substances; sensitivity increasing substances such as polyethylene glycol; nonspecific reaction inhibiting substances; or nonionic surfactants, amphoteric surfactants or anions You may make it coexist with 1 type, or 2 or more types, such as various surfactants, such as an ionic surfactant.
In the measuring method and measuring reagent of the present invention, a specific binding substance that binds to CRP in a calcium ion-dependent manner and a carrier on which a specific binding substance that binds to CRP in a calcium ion-independent manner is immobilized, It is preferable to coexist with ions or calcium salts.
The concentration when the above substances coexist is not particularly limited, but is preferably 0.001 to 10% (W / V), and particularly preferably 0.01 to 5% (W / V). .
[8] Sample
In the present invention, the term “sample” refers to a sample in which CRP may be present and the presence or absence or content (concentration) of CRP is to be measured.
Examples of such samples include body fluids such as human or animal blood, serum, plasma, spinal fluid or ascites, or extracts such as organs, tissues or cells, which may contain CRP. be able to.
[9] Container
The container used for the method for measuring CRP in the sample of the present invention is not particularly limited, and a suitable one can be used.
[10] Measuring reagent
The reagent for measuring CRP in a sample according to the present invention contains a specific binding substance that binds to CRP in a calcium ion-dependent manner and a carrier on which a specific binding substance that binds to CRP in a calcium ion-independent manner is immobilized. It is characterized by.
As a result, the measurement range of CRP in a sample can be expanded from a low concentration to a high concentration, and a wide range of CRP from a low concentration to a high concentration can be accurately measured.
The measurement reagent of the present invention may consist of one measurement reagent.
In this case, a specific binding substance that binds to CRP in a calcium ion-dependent manner and a carrier on which a specific binding substance that binds to CRP in a calcium ion-independent manner is immobilized is contained in one of the measurement reagents.
The measurement reagent of the present invention may be composed of two or more measurement reagents.
In this case, a specific binding substance that binds to CRP in a calcium ion-dependent manner and a carrier on which a specific binding substance that binds to CRP in a calcium ion-independent manner is immobilized is one of two or more measurement reagents. It may be contained in a measurement reagent, or may be contained in two or more measurement reagents.
For example, when the measurement reagent of the present invention is composed of two measurement reagents, a first reagent and a second reagent, a specific binding substance that binds to CRP in a calcium ion-dependent manner, and calcium ion-independent The carrier on which the specific binding substance that binds to CRP is immobilized may be contained only in the first reagent, may be contained only in the second reagent, and further, the first reagent and the second reagent. You may make it contain in both.
When the measurement reagent of the present invention comprises two measurement reagents, a specific binding substance that binds to CRP in a calcium ion-dependent manner and a carrier on which a specific binding substance that binds to CRP in a calcium ion-independent manner is immobilized Is preferably contained only in the second reagent.
When the measurement reagent of the present invention is composed of two or more measurement reagents, a specific binding substance that binds to CRP in a calcium ion-dependent manner and a specific binding substance that binds to CRP in a calcium ion-independent manner. A reagent other than a reagent containing a carrier on which a binding substance is immobilized, that is, a specific binding substance that binds to CRP in a calcium ion-dependent manner and a specific binding substance that binds to CRP in a calcium ion-independent manner are immobilized. The reagent not containing a carrier may be, for example, a buffer solution.
In addition, various aqueous solvents can be used as a solvent for the measurement reagent of the present invention.
Examples of the aqueous solvent include purified water, physiological saline, or various buffer solutions such as tris (hydroxymethyl) aminomethane buffer, phosphate buffer, or phosphate buffered saline. .
The pH of the buffer solution may be appropriately selected and used as appropriate. Although there is no particular limitation, it is general to select and use a pH within the range of pH 5 to pH 10.
Further, the specific binding substance that binds to CRP in a calcium ion-dependent manner and the specific binding substance that binds to CRP in a calcium ion-independent manner are immobilized on the CRP measurement reagent in the sample of the present invention. In addition to carriers, proteins such as bovine serum albumin (BSA), human serum albumin (HSA), casein or salts thereof; various metal ions such as calcium ions; various salts such as calcium salts; various sugars; skim milk powder; normal rabbits Various animal sera such as serum; various preservatives such as sodium azide or antibiotics; activating substances; reaction promoting substances; sensitivity increasing substances such as polyethylene glycol; nonspecific reaction inhibiting substances; or nonionic surfactants , One or more of various surfactants such as amphoteric surfactant or anionic surfactant It may be contained as appropriate.
The measurement reagent of the present invention preferably contains calcium ions or calcium salts.
The concentration when these are contained in the measurement reagent of the present invention is not particularly limited, but is preferably 0.001 to 10% (W / V), particularly 0.01 to 5% (W / V). ) Is preferred.
Examples of the surfactant include sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, Nonionic surfactants such as polyoxyethylene phytosterol, phytostanol, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil or polyoxyethylene lanolin; betaine acetate, etc. Amphoteric surfactants; or polyoxyethylene alkyl ether sulfate or polyoxyethylene alkyl ether acetic acid Anionic surfactants, such as and the like.
The reagent for measuring CRP in the sample of the present invention can be sold alone or used for measuring CRP in the sample.
In addition, the CRP measuring reagent in the sample of the present invention can be sold in combination with other reagents or used for measuring CRP in the sample.
Examples of the other reagent include a buffer solution, a sample diluent, a reagent diluent, a reagent containing a labeling substance, a reagent containing a substance that generates a signal such as color development, or calibration (calibration). And reagents containing these substances.
[11] Measuring method
In the method for measuring CRP in a sample according to the present invention, a specific binding substance that binds to CRP in a calcium ion-dependent manner and a specific binding substance that binds to CRP in a calcium ion-independent manner are immobilized on a carrier, respectively. CRP in the sample is measured by measuring a complex aggregate of the specific binding substance and CRP generated by the specific binding reaction between the specific binding substance immobilized on the CRP and the CRP contained in the sample. Is a method of measuring.
The measurement operation in the method for measuring CRP in the sample of the present invention can be performed according to a known measurement operation.
For example, a specific binding substance that binds to CRP in a calcium ion-dependent manner and a carrier on which a specific binding substance that binds to CRP in a calcium ion-independent manner is immobilized [for example, “binding to CRP in a calcium ion-dependent manner” A mixture of a carrier having a specific binding substance immobilized thereon and a carrier having a specific binding substance immobilized on CRP in a calcium ion-independent manner, or “specific binding binding to CRP in a calcium ion-dependent manner” A measurement reagent containing a substance and a specific binding substance that binds to CRP in a calcium ion-independent manner immobilized on the same carrier is prepared and prepared.
Next, the sample is mixed with a specific binding substance that binds to CRP in a calcium ion-dependent manner and a measurement reagent containing a carrier on which a specific binding substance that binds to CRP in a calcium ion-independent manner is immobilized, Make contact.
Thus, a specific binding substance that binds to CRP in a calcium ion-dependent manner immobilized on a carrier, a specific binding substance that binds to CRP in a calcium ion-independent manner, and the CRP contained in the sample, A specific binding reaction is performed.
In addition, when performing this specific binding reaction, it is preferable to make calcium ion or a calcium salt exist.
Next, a specific binding substance that binds to CRP in a calcium ion-dependent manner immobilized on the carrier, and a specific binding substance that binds to CRP in a calcium ion-independent manner, which are generated by this specific binding reaction, Measure complex aggregates.
The measurement of the generated complex aggregate should be performed by measuring the absorbance of the reaction mixture, such as transmitted light or scattered light, in the measurement reaction in which the complex aggregate is present by the endpoint method or the rate method. To implement.
Then, the measured value such as the absorbance obtained by measuring the sample is compared with the measured value such as the absorbance obtained by measuring the standard substance (sample having a known CRP concentration), and the CRP contained in the sample is measured. The concentration (quantitative value) of is calculated.
The measurement of absorbance such as transmitted light or scattered light may be performed by measuring transmitted light or scattered light, and may be one-wavelength measurement or two-wavelength measurement (two It may be a difference or ratio depending on the wavelength.
The measurement wavelength is generally selected from 340 nm to 1,000 nm.
In addition, the measurement of CRP in the sample of the present invention may be performed by a method, or may be performed using an apparatus such as a measuring apparatus.
The measuring device may be a general-purpose automatic analyzer or a dedicated measuring device (dedicated machine).
In addition, this measurement may be performed by a one-step method (one-reagent method) or by a method performed by a plurality of operation steps such as a two-step method (two-reagent method).
Hereinafter, a specific description will be given, taking as an example the case of measuring CRP in a sample using a CRP measurement reagent in the sample using the latex turbidimetry as a measurement principle.
(1) First, the following are prepared and prepared as measurement reagents for CRP in a sample.
First reagent: Buffer solution containing a buffer and adjusting the pH to a constant value
Second reagent: Buffer containing an antibody that binds to CRP in a calcium ion-dependent manner and an antibody that binds to CRP in a calcium ion-independent manner immobilized on the same latex particle
(2) A certain amount of a sample such as serum and a certain amount of the first reagent are mixed and allowed to stand at a certain temperature for a certain time.
In addition, what is necessary is just to select the mixing ratio (quantity ratio) of a sample and a 1st reagent suitably.
Further, the temperature at the time of standing is preferably a constant temperature within a range of room temperature (1 ° C. to 30 ° C.) or slightly warm (30 ° C. to 40 ° C.). (For example, 37 ° C.)
(3) After a certain time, a certain amount of the second reagent is added to and mixed with the mixed solution of the sample and the first reagent, and left as a reaction mixture at a constant temperature for a certain time.
In addition, what is necessary is just to select the addition amount of a 2nd reagent suitably.
Further, the temperature at the time of standing is preferably a constant temperature within a range of room temperature (1 ° C. to 30 ° C.) or slightly warm (30 ° C. to 40 ° C.). (For example, 37 ° C.)
The standing time is preferably a fixed time of 1 minute or more and 10 minutes or less, and more preferably a fixed time of 3 minutes or more and 5 minutes or less.
An antibody that binds to CRP in a calcium ion-dependent manner immobilized on latex particles by adding a second reagent to the mixed solution of the sample and the first reagent, and an antibody that binds to CRP in a calcium ion-independent manner; Then, an antigen-antibody reaction (measurement reaction) with CRP contained in the sample is performed.
Then, by this antigen-antibody reaction (measurement reaction), "... [antibody binding to CRP dependent on calcium ion = latex particle = antibody binding to CRP independent of calcium ion]-[CRP]-[calcium ion dependent] An antibody that specifically binds to CRP = latex particles = an antibody that binds to CRP in a calcium ion-independent manner], etc. ”, and the like. Complex aggregates of “latex particles immobilized with antibodies that bind to CRP in a calcium ion-independent manner” are formed.
(4) Then, in an analyzer or spectrophotometer, the reaction mixture is irradiated with light, and a decrease in transmitted light intensity at an appropriate wavelength, which is a signal generated by a composite aggregate of latex particles generated (absorbance) ) Or an increase in scattered light intensity, the amount of the complex aggregate produced, that is, the amount of CRP contained in the sample is determined.
(5) And "measured value obtained by measuring the sample (decrease in transmitted light intensity (increase in absorbance) or increase in scattered light intensity)" and "standard substance such as standard solution or standard serum ( Sample measured by comparing with the measured value (decrease in transmitted light intensity (increase in absorbance) or increase in scattered light intensity) obtained by measuring CRP with a known concentration) The amount (concentration) of CRP contained therein is calculated.
EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by these Examples.

(Confirmation of anti-CRP antibody)
Using a peroxidase-labeled antibody and a CRP-immobilized plate, each of the four anti-CRP antibodies is an antibody that binds to CRP in a calcium ion-dependent manner by the sandwich method of ELISA (enzyme immunoassay), or It was determined whether the antibody binds to CRP in a calcium ion-independent manner.
1. Preparation of Reagent (1) Preparation of Peroxidase-Labeled Antibody A peroxidase-labeled goat anti-mouse IgG antibody solution (Jacson ImmunoResearch Laboratories) was diluted 2000 times with a 0.5% casein solution to prepare a peroxidase-labeled antibody.
(2) Preparation of Washing Solution Phosphate buffered saline [pH 7.2 (20 ° C.)] containing 0.05% Tween 20 (Tween 20) was prepared and used as a washing solution.
(3) Preparation of CRP-immobilized plate The recombinant CRP (Oriental Yeast Co., Ltd.) was diluted with 10 mM phosphate buffered saline [pH 7.4 (20 ° C.)] to a concentration of 5 μg / mL, 50 μL per well was added to a 96-well microtiter plate (NUNK) and allowed to stand at 37 ° C. for 2 hours to adsorb and immobilize the CRP in each well of the microplate.
The microtiter plate on which the CRP is immobilized is washed by adding 300 μL of the washing solution (2) per well, and this washing is performed 5 times in total, and then 0.5% containing 0.05% sodium azide. % Casein solution was added at 200 μL per well and allowed to stand at 4 ° C. overnight for blocking treatment to prepare a CRP-immobilized plate.
(4) Preparation of Coloring Solution To 100 mL of 0.1 M citrate buffer [pH 4.0 (20 ° C.)], ABTS (2,2′-azinobis [3-ethyl-2,3-dihydrobenzothiazole-6- Sulfonic acid]) (Roche Diagnostics Co., Ltd.) 50 mg and 30% hydrogen peroxide water 10 μL were added immediately before use to prepare a coloring solution.
2. Preparation of Antibody Sample (1) Preparation of Diluent A Calcium chloride was dissolved in a 0.5% casein aqueous solution so as to have a concentration of 2 mM.
(2) Preparation of Diluent B Prepared by dissolving EDTA.2 sodium salt (Dojindo Laboratories), which is a chelating agent, in a 0.5% casein aqueous solution so as to have a concentration of 10 mM. did.
(3) Anti-CRP antibodies The following four types of commercially available antibodies were prepared as anti-CRP antibodies.
(A) Antibody-1: Mouse anti-human CRP monoclonal antibody [clone: CRB-018] (Japan Biotest Laboratories)
(B) Antibody-2: Mouse anti-human CRP monoclonal antibody [clone: CRB-023] (Japan Biotest Laboratories)
(C) Antibody-3: Mouse anti-human CRP monoclonal antibody [clone: CRB-031] (Japan Biotest Laboratories)
(D) Antibody-4: Mouse anti-human CRP monoclonal antibody [clone: CRB-032] (Japan Biotest Laboratories)
(4) Preparation of antibody sample (a) Calcium ion-containing anti-CRP antibody sample The four types of anti-CRP antibodies from antibody-1 to antibody-4 of (3) above were prepared in the diluent A prepared in (1) above. Each containing a calcium ion-containing anti-CRP having an anti-CRP antibody concentration of 0.010 μg / mL, 0.10 μg / mL, 1.0 μg / mL, 10.0 μg / mL, or 100.0 μg / mL, respectively. Each antibody sample was prepared.
Further, the diluent A prepared in the above (1) was used as a calcium ion-containing anti-CRP antibody sample having an anti-CRP antibody concentration of 0 μg / mL.
(B) EDTA-containing / anti-CRP antibody sample The four types of anti-CRP antibodies from antibody-1 to antibody-4 in (3) above were each diluted with the diluent B prepared in (2) above to obtain anti-CRP antibodies. EDTA-containing / anti-CRP antibody samples having concentrations of 0.010 μg / mL, 0.10 μg / mL, 1.0 μg / mL, 10.0 μg / mL, or 100.0 μg / mL, respectively, were prepared.
Further, the diluent B prepared in the above (2) was used as an EDTA-containing anti-CRP antibody sample having an anti-CRP antibody concentration of 0 μg / mL.
3. Measurement by ELISA method (1) Measurement (b) Washing was performed by adding 400 μL of the washing solution prepared in (2) above to each well of the CRP-immobilized plate prepared in (1) above. This washing was performed 5 times in total.
(B) Thereafter, for each of the four types of anti-CRP antibodies from antibody-1 to antibody-4 prepared in (2) of (4) above, the concentration of anti-CRP antibody was 0 μg / mL, 0. 010 μg / mL, 0.10 μg / mL, 1.0 μg / mL, 10.0 μg / mL, or 100.0 μg / mL calcium ion-containing anti-CRP antibody sample [anti-CRP antibody 4 types × 6 concentration = total 24 50 μL of each type) was added to each well of the CRP-immobilized plate washed in (a), and allowed to stand at 37 ° C. for 2 hours, and the CRP immobilized on each well of the CRP-immobilized plate; The antigen-antibody reaction with each anti-CRP antibody was performed.
At this time, the CRP immobilized in each well of the CRP-immobilized plate becomes calcium-binding CRP by adding and contacting the calcium ion-containing / anti-CRP antibody sample.
Therefore, at this time, the binding property of each anti-CRP antibody to the calcium-binding CRP immobilized in each well is tested.
(C) Thereafter, the calcium ion-containing anti-CRP antibody sample in each well of the CRP-immobilized plate is removed by aspiration, and the washing solution prepared in (1) above is added to each well in an amount of 400 μL per well. This was washed a total of 5 times.
(D) Next, 50 μL of the peroxidase-labeled antibody prepared in (1) above was added to each well of the CRP-immobilized plate and reacted at 37 ° C. for 1 hour.
(E) Next, each well of the CRP-immobilized plate was washed by adding 400 μL of the washing solution prepared in (1) above per well, and this washing was performed 5 times in total.
(F) Thereafter, 100 μL of the color developing solution prepared in (1) above was added to each well of the CRP-immobilized plate and allowed to react at room temperature for 30 minutes.
(G) Thereafter, the absorbance at 415 nm of the reaction solution in each well of the CRP-immobilized plate was measured using a microplate reader (BioRad; Model 3550).
(H) The anti-CRP antibody concentrations are 0.010 μg / mL, 0.10 μg / mL, 1.0 μg / mL, and 10.0 μg / mL for each of the four types of anti-CRP antibodies from antibody-1 to antibody-4. Alternatively, the measurement value (absorbance) of the calcium ion-containing anti-CRP antibody sample with 100.0 μg / mL is subtracted from the measurement value (absorbance) of the calcium ion-containing anti-CRP antibody sample with an anti-CRP antibody concentration of 0 μg / mL. Then, the value of the absorbance difference was determined.
(I) In place of (b), EDTA-containing anti-CRP antibody sample [anti-CRP antibody 4 types × 6] instead of calcium ion-containing anti-CRP antibody sample (anti-CRP antibody 4 types × 6 concentration = total 24 types) The value of the absorbance difference was determined by performing the operations as described in the above (a) to (h) except that 50 μL (concentration = total 24 types) was added to each well of the CRP-immobilized plate. .
In this measurement of the EDTA-containing / anti-CRP antibody sample, the calcium ions bound to the CRP immobilized in each well of the CRP-immobilized plate by the addition and contact of the EDTA-containing / anti-CRP antibody sample were: The CRP immobilized in each of the wells by binding with EDTA · 2Na is a calcium non-binding CRP.
Therefore, at this time, the binding property of each anti-CRP antibody to the calcium non-binding CRP immobilized in each well is tested.
(2) Measurement results In the above (1), calcium ion-containing anti-CRP antibody samples (4 types of anti-CRP antibodies × 6 concentrations = total 24 types) and EDTA-containing anti-CRP antibody samples [4 types of anti-CRP antibodies × 6 Concentrations = total 24 types) were measured, and the absorbance difference values obtained in the test of the binding of each anti-CRP antibody to calcium-bound CRP and calcium-unbound CRP are shown in Table 1 (antibody- 1 to antibody-4), and FIG. 1 (antibody-1), FIG. 2 (antibody-2), FIG. 3 (antibody-3) and FIG. 4 (antibody-4).

In FIGS. 1, 2, 3 and 4, the horizontal axis represents the concentration (μg / mL) of the anti-CRP antibody in the antibody sample added to the well of the CRP-immobilized plate, and the vertical axis represents the measurement. The obtained absorbance difference value (415 nm) is represented.
In FIG. 1, FIG. 2, FIG. 3 and FIG. 4, “●” indicates the measurement result (absorbance difference) when each calcium ion-containing / anti-CRP antibody sample is measured, and “▲” indicates each The measurement results (absorbance difference) when an EDTA-containing anti-CRP antibody sample is measured are shown.
4). Discussion (a) From the above examination results, antibody-1 (FIG. 1), antibody-2 (FIG. 2), and antibody-3 (FIG. 3) each bind to calcium-binding CRP of each anti-CRP antibody. Curves of measurement results (calcium ion-containing / anti-CRP antibody samples) showing sex and measurement results (EDTA-containing / anti-CRP antibody samples) showing the binding properties of each anti-CRP antibody to calcium non-binding CRP, You can see that they are far apart.
That is, it can be seen that antibody-1, antibody-2, and antibody-3 have significantly lower binding to calcium-unbound CRP than binding to calcium-bound CRP.
From this, it can be determined that each of antibody-1, antibody-2, and antibody-3 is a “specific binding substance (antibody) that binds to CRP in a calcium ion-dependent manner”.
(B) In antibody-4 (FIG. 4), the curve of the measurement results (calcium ion-containing anti-CRP antibody sample) showing the binding property of the anti-CRP antibody to the calcium-binding CRP and the calcium of the anti-CRP antibody It can be seen that the curves of the measurement results (EDTA-containing anti-CRP antibody sample) showing the binding property to the non-binding CRP are hardly separated.
That is, it can be seen that antibody-4 binds to approximately the same extent as both calcium-bound CRP and calcium-unbound CRP.
From this, it can be determined that the antibody-4 is a “specific binding substance (antibody) that binds to CRP independent of calcium ions”.

(Confirmation of latex particles immobilized with anti-CRP antibody)
Reactivity with CRP in the sample was confirmed for latex particles immobilized with an antibody that binds to CRP in a calcium ion-dependent manner, or latex particles immobilized with an antibody that binds to CRP in a calcium ion-independent manner.
1. Reagent (1) 1st reagent 100% tris (hydroxymethyl) aminomethane containing 1% (w / v) BSA, 2M sodium chloride, 0.2mM calcium chloride dihydrate and 0.05% sodium azide -A hydrochloric acid buffer (pH 8.0 (20 ° C)) was prepared and used as the first reagent.
(2) Second reagent (a) Second reagent [antibody-1]
Antibody (1) in Example 1, 2 (3) -1 [specific binding substance (antibody) that binds to CRP dependently on calcium ion) in 0.094 mL of a 10% suspension of latex particles having an average particle size of 0.1 μm ] In a concentration of 0.1 g / dL in 6.7 mM MES buffer [pH 5.0 (20 ° C.)] was added, and the mixture was stirred at 5 ° C. overnight. Thereby, the antibody-1 was immobilized on latex particles.
Next, after removing the supernatant by centrifugation, 100 mM glycine buffer solution [pH 9.5 (20 ° C.)] containing 1.0% bovine serum albumin (BSA) is added to the precipitate and suspended therein. Stir for a minute and perform blocking treatment.
Next, the precipitate was recovered by centrifugation, and then suspended in a 0.05% aqueous sodium azide solution so that the absorbance at a wavelength of 585 nm was 9.5 OD.
Then, this was diluted with a 0.05% aqueous sodium azide solution to prepare a 0.060% suspension of latex particles on which antibody-1 was immobilized.
This was designated as a second reagent [antibody-1].
(B) Second reagent [antibody-2]
Except that the antibody-1 in (a) is changed to the antibody-2 (specific binding substance (antibody) that binds to CRP in a calcium ion-dependent manner) in 2 (3) of Example 1, ) To prepare a 0.060% suspension of latex particles on which antibody-2 is immobilized.
This was designated as a second reagent [antibody-2].
(C) Second reagent [antibody-3]
Except for changing the antibody-1 in (a) to the antibody-3 [specific binding substance (antibody) that binds to CRP in a calcium ion-dependent manner] in 2 (3) of Example 1, ) To prepare a 0.060% suspension of latex particles with antibody-3 immobilized thereon.
This was designated as a second reagent [antibody-3].
(D) Second reagent [antibody-4]
Except that the antibody-1 in (a) is changed to the antibody-4 [specific binding substance (antibody) that binds to CRP in a calcium ion-independent manner) in 2 (3) of Example 1, The operation was performed as described in (a) to prepare a 0.060% suspension of latex particles on which antibody-4 was immobilized.
This was designated as a second reagent [antibody-4].
2. Sample (1) Preparation of Sample Diluent 50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid containing 4% (w / v) BSA, 0.2 mM calcium chloride dihydrate, 100 mM sodium chloride and 15 mM sodium azide A buffer solution [pH 7.6] was prepared and used as a sample diluent.
(2) Preparation of sample Each sample of the following CRP concentration was prepared by diluting genetically modified CRP (manufactured by Oriental Yeast Co., Ltd.) with the sample diluent of (1) above.
Further, physiological saline (0.9% sodium chloride aqueous solution) was used as a sample having a CRP concentration of 0 mg / dL.
(A) Sample 1: 0 mg / dL
(B) Sample 2: 0.5 mg / dL
(C) Sample 3: 2.0 mg / dL
(D) Sample 4: 6.0 mg / dL
(E) Sample 5: 18.0 mg / dL
(F) Sample 6: 30.0 mg / dL
3. Measurement of CRP in sample (1) Measurement procedure (A) Measurement was performed using a Hitachi-7180 type automatic analyzer (manufactured by Hitachi, Ltd.).
First, 3 μL of Sample 1, Sample 2, Sample 3, Sample 4, Sample 5, or Sample 6 of (2) above was added to a measurement cell (cuvette).
Next, 100 μL of the first reagent (1) was added to these measurement cells (cuvettes) and mixed.
These measurement cells (cuvettes) were allowed to stand at 37 ° C.
(B) At 4 minutes and 34 seconds (16th point) after the addition of the first reagent, the mixed liquid in these measurement cells (cuvette) is further added to the item (1) in (1) (2) above. 100 μL of the second reagent [antibody-1] was added and mixed.
(C) At 5 minutes 09 seconds (18th point) after the addition of the first reagent, the absorbance of the liquid mixture (main wavelength 570 nm, subwavelength 800 nm) in these measurement cells (cuvette) was blinded to the sample. As measured.
These measurement cells (cuvettes) were allowed to stand at 37 ° C. for reaction.
As a result, an antigen-antibody reaction between the anti-CRP antibody immobilized on the latex particles and the CRP contained in the sample was performed to generate latex particle aggregates.
(D) At 9 minutes and 54 seconds (34 points) after the addition of the first reagent, the absorbance (main wavelength 570 nm, subwavelength 800 nm) of the reaction mixture in this measurement cell (cuvette) It was measured as a measured value.
(E) The absorbance (sample blind) measured in (b) was subtracted from the absorbance (measured value) measured in (d) above to obtain an absorbance difference.
This difference in absorbance is proportional to the amount (concentration) of CRP contained in the sample.
(F) In addition, in (b) above, except that the second reagent [antibody-2] of (b) in (1) above is used instead of the second reagent [antibody-1] ( The operations were carried out as in (a) to (e), and the absorbance difference of each sample was obtained when the second reagent [antibody-2] was used as the second reagent.
(G) In addition, in (b) above, except that the second reagent [antibody-3] of (1) (2) (c) is used instead of the second reagent [antibody-1] ( The operations were carried out as in (a) to (e), and the absorbance difference of each sample was obtained when the second reagent [antibody-3] was used as the second reagent.
(H) In (b) above, except that the second reagent [antibody-4] of (1) above (2) is used instead of the second reagent [antibody-1] The operations were carried out as in (a) to (e), and the absorbance difference of each sample was obtained when the second reagent [antibody-4] was used as the second reagent.
(2) Measurement results Table 2 and Fig. 5 show the absorbance difference obtained by measuring CRP in the sample in (1).
In FIG. 5, the horizontal axis represents the CRP concentration (mg / dL) in the sample, and the vertical axis represents the absorbance difference value obtained by measurement [absorbance difference × 10,000] (main wavelength 570 nm, subwavelength 800 nm. ).
In FIG. 5, “□” indicates the measurement result (absorbance difference value) when “second reagent [antibody-1]” is used as the second reagent, and “◯” indicates “ The measurement results (absorbance difference value) when using the second reagent [antibody-2] are shown, and “Δ” is the measurement result when using the second reagent [antibody-3] as the second reagent. (Absorbance difference value), and “◇” indicates a measurement result (absorbance difference value) when “second reagent [antibody-4]” is used as the second reagent.

4). Discussion (a) From Table 2 and FIG. 5, the anti-CRP antibody of “carrier (latex particles) on which anti-CRP antibody is immobilized” to be contained in the CRP measurement reagent in the sample is expressed as “calcium ion dependent CRP. When the above-mentioned “antibody-1”, “antibody-2”, and “antibody-3” which are “specific binding substances (antibodies)” are used, CRP in the sample is in a low concentration range. Sometimes the reactivity with CRP is low and the sensitivity of the measurement is not high, but the reactivity with CRP is high from the medium concentration range to the high concentration range, and the value of the absorbance difference increases as the CRP concentration in the sample increases. It can be seen that the amount of complex aggregates increases as the CRP concentration in the sample increases.
Therefore, when “antibody-1”, “antibody-2”, and “antibody-3” are used, that is, “specific binding substance (antibody) that binds to CRP in a calcium ion-dependent manner” is used. However, it was confirmed that although the sensitivity was low in the low concentration range, the sensitivity was high and the measurement was quantitative from the middle concentration range to the high concentration range.
(B) From Table 2 and FIG. 5, the anti-CRP antibody of the “carrier (latex particle) on which the anti-CRP antibody is immobilized” to be contained in the CRP measurement reagent in the sample is “calcium ion-independent. When the above-mentioned “antibody-4” which is a “specific binding substance (antibody) that binds to CRP” is used, when the CRP in the sample is in a low concentration range, the reactivity with CRP is high and the sensitivity of the measurement However, when the CRP concentration in the sample increases, the absorbance difference value does not increase or decreases from the middle concentration range to the high concentration range, that is, even if the CRP concentration in the sample increases. It can be seen that the amount of body aggregates produced does not increase or decreases.
Therefore, when the above-mentioned “antibody-4” is used, that is, when “specific binding substance (antibody) that binds to CRP independent of calcium ion” is used, it is highly sensitive in a low concentration range. In addition, it was confirmed that the measurement was not quantitative in the middle concentration range to the high concentration range.

(Measuring method of CRP in sample of the present invention and confirmation of effect of measuring reagent-1)
The present invention comprising latex particles immobilized with an antibody (antibody-1) that binds to CRP in a calcium ion-dependent manner and latex particles immobilized with an antibody (antibody-4) that binds to a CRP in a calcium ion-independent manner. The CRP in the sample was measured using the CRP measurement reagent, and the effect was confirmed.
1. Reagent (1) 1st reagent 100% tris (hydroxymethyl) aminomethane containing 1% (w / v) BSA, 2M sodium chloride, 0.2mM calcium chloride dihydrate and 0.05% sodium azide -A hydrochloric acid buffer (pH 8.0 (20 ° C)) was prepared and used as the first reagent.
(2) Preparation of Second Reagent (a) Antibody-1 Immobilized Latex Particle Suspension To 0.094 mL of 10% suspension of latex particles having an average particle size of 0.1 μm, (2) of Example 1-2 Antibody-1 [specific binding substance (antibody) that binds to CRP in a calcium ion-dependent manner] mixed with 6.7 mM MES buffer (pH 5.0 (20 ° C.)) at a concentration of 0.1 g / dL 0.5098 mL was added and it stirred at 5 degreeC overnight.
Next, after removing the supernatant by centrifugation, 100 mM glycine buffer solution (pH 9.5 (20 ° C.) containing 1.0% BSA) is added to the precipitate and suspended, and the mixture is stirred at room temperature for 30 minutes to block. Processed.
Next, the precipitate was recovered by centrifugation, and then suspended in a 0.05% aqueous sodium azide solution so that the absorbance at a wavelength of 585 nm was 9.5 OD.
This was diluted with 0.05% aqueous sodium azide solution to prepare a 0.120% suspension of latex particles on which antibody-1 (an antibody that binds to CRP in a calcium ion-dependent manner) was immobilized.
This was designated as antibody-1 immobilized latex particle suspension.
(B) Preparation of Antibody-4 Immobilized Latex Particle Suspension To 0.094 mL of a 10% suspension of latex particles having an average particle size of 0.1 μm, antibody-4 [calcium of Example 1, 2 (3) was added. 0.5098 mL of a solution in which a specific binding substance (antibody) that binds to CRP in an ion-independent manner is mixed with 6.7 mM MES buffer (pH 5.0 (20 ° C.)) at a concentration of 0.1 g / dL is added. Stir at 5 ° C. overnight.
Next, after removing the supernatant by centrifugation, 100 mM glycine buffer solution (pH 9.5 (20 ° C.) containing 1.0% BSA) is added to the precipitate and suspended, and the mixture is stirred at room temperature for 30 minutes to block. Processed.
Next, the precipitate was recovered by centrifugation, and then suspended in a 0.05% aqueous sodium azide solution so that the absorbance at a wavelength of 585 nm was 9.5 OD.
This was diluted with 0.05% aqueous sodium azide solution to prepare a 0.036% suspension of latex particles on which antibody-4 (an antibody that binds to CRP in a calcium ion-independent manner) was immobilized. .
This was designated as antibody-4 immobilized latex particle suspension.
(C) Preparation of Second Reagent [Antibody-1] 5 mL of the antibody-1 immobilized latex particle suspension prepared in (a) above and 5 mL of 0.05% sodium azide aqueous solution were mixed. A suspension containing 0.060% of “antibody-1 (an antibody that binds to CRP in a calcium ion-dependent manner)” was prepared.
This was designated as a second reagent [antibody-1].
(D) Preparation of second reagent [antibody-4] 5 mL of the antibody-4-immobilized latex particle suspension prepared in (b) above and 5 mL of 0.05% aqueous sodium azide solution were mixed. A suspension containing 0.018% of “antibody-4 (an antibody that binds to CRP in a calcium ion-independent manner)” was prepared.
This was designated as a second reagent [antibody-4].
(E) Preparation of Second Reagent [Antibody-1 / Antibody-4] 5 mL of the antibody-1 immobilized latex particle suspension prepared in (a) above, and Antibody-4 immobilization prepared in (b) above. 5 mL of the latex particle suspension was mixed with 0.060% of “antibody-1 (an antibody that binds to CRP in a calcium ion-dependent manner)” and 0.018% of “antibody— A suspension containing “latex particles immobilizing 4 (an antibody that binds to CRP in a calcium ion-independent manner)” was prepared.
This was designated as a second reagent [antibody-1 / antibody-4].
2. Sample (1) Preparation of Sample Diluent 50 mM Tris (hydroxymethyl) aminomethane-hydrochloric acid containing 4% (w / v) BSA, 0.2 mM calcium chloride dihydrate, 100 mM sodium chloride and 15 mM sodium azide A buffer solution [pH 7.6] was prepared and used as a sample diluent.
(2) Preparation of sample Each sample of the following CRP concentration was prepared by diluting genetically modified CRP (manufactured by Oriental Yeast Co., Ltd.) with the sample diluent of (1) above.
Further, physiological saline (0.9% sodium chloride aqueous solution) was used as a sample having a CRP concentration of 0 mg / dL.
(A) Sample 1: 0 mg / dL
(B) Sample 2: 0.5 mg / dL
(C) Sample 3: 2.0 mg / dL
(D) Sample 4: 6.0 mg / dL
(E) Sample 5: 18.0 mg / dL
(F) Sample 6: 30.0 mg / dL
3. Measurement of CRP in sample (1) Measurement procedure (A) Measurement was performed using a Hitachi-7180 type automatic analyzer (manufactured by Hitachi, Ltd.).
First, 3 μL of Sample 1, Sample 2, Sample 3, Sample 4, Sample 5, or Sample 6 of (2) above was added to a measurement cell (cuvette).
Next, 100 μL of the first reagent (1) was added to these measurement cells (cuvettes) and mixed.
These measurement cells (cuvettes) were allowed to stand at 37 ° C.
(B) At 4 minutes and 34 seconds (16th point) after the addition of the first reagent, the liquid mixture in these measurement cells (cuvette) is further added to the (1) (2) (c). 100 μL of the second reagent [antibody-1] was added and mixed.
(C) At 5 minutes 09 seconds (18th point) after the addition of the first reagent, the absorbance of the liquid mixture (main wavelength 570 nm, subwavelength 800 nm) in these measurement cells (cuvette) was blinded to the sample. As measured.
These measurement cells (cuvettes) were allowed to stand at 37 ° C. for reaction.
As a result, an antigen-antibody reaction between the anti-CRP antibody immobilized on the latex particles and the CRP contained in the sample was performed to generate latex particle aggregates.
(D) At 9 minutes and 54 seconds (34 points) after the addition of the first reagent, the absorbance (main wavelength 570 nm, subwavelength 800 nm) of the reaction mixture in this measurement cell (cuvette) It was measured as a measured value.
(E) The absorbance (sample blind) measured in (c) above was subtracted from the absorbance (measured value) measured in (d) above to obtain an absorbance difference.
This difference in absorbance is proportional to the amount (concentration) of CRP contained in the sample.
(F) In addition, in (b), in place of the second reagent [antibody-1], except that the second reagent [antibody-4] of (1) in (2) above is used, The operations were carried out as in (a) to (e) above, and the absorbance difference of each sample was obtained when the second reagent [antibody-4] was used as the second reagent.
(G) Also, in (b) above, the second reagent [antibody-1 / antibody-4] of (e) in (1) above is used in place of the second reagent [antibody-1]. Except for the above, operations were carried out as in (a) to (e) above, and the absorbance difference of each sample was obtained when the second reagent [antibody-1 / antibody-4] was used as the second reagent.
(2) Measurement result Table 3 and FIG. 6 show the absorbance difference obtained by measuring CRP in the sample in (1).
In FIG. 6, the horizontal axis represents the CRP concentration (mg / dL) in the sample, and the vertical axis represents the absorbance difference value obtained by measurement [absorbance difference × 10,000] (main wavelength 570 nm, subwavelength 800 nm. ).
In FIG. 6, “Δ” indicates the measurement result (absorbance difference value) when “second reagent [antibody-1]” is used as the second reagent, and “◯” indicates “ The measurement results (absorbance difference values) when using the second reagent [antibody-4] are shown, and “■” uses “second reagent [antibody-1 / antibody-4]” as the second reagent. The measurement results (absorbance difference values) are shown.

4). Discussion (a) From Table 3 and FIG. 6, as an anti-CRP antibody of “a carrier (latex particle) on which an anti-CRP antibody is immobilized” contained in a CRP measurement reagent in a sample, When the above-mentioned “antibody-1” which is a “specific binding substance (antibody) to be bound” is used (when the second reagent is “second reagent [antibody-1]”), It can be seen that when CRP is in the low concentration range, the sensitivity is low, but the sensitivity is high in the medium concentration range to the high concentration range and has a quantitative property of measurement.
(B) From Table 3 and FIG. 6, the “antibody-4”, which is a “specific binding substance (antibody) that binds to CRP independent of calcium ions”, is used as the anti-CRP antibody. If the second reagent is “second reagent [antibody-4]”, the CRP in the sample is highly sensitive when it is in the low concentration range, but the medium concentration range to the high concentration range. It can be seen that the measurement does not have the quantitativeness of measurement.
(C) On the other hand, from Table 3 and FIG. 6, as an anti-CRP antibody of “a carrier (latex particles) on which an anti-CRP antibody is immobilized” contained in a CRP measurement reagent in a sample, The above-mentioned “antibody-1” which is a specific binding substance (antibody) that binds to CRP ”and the“ antibody ”which is a“ specific binding substance (antibody) that binds to CRP independently of calcium ions ” -4 "(when the second reagent is" second reagent [antibody-1 / antibody-4] "), the CRP in the sample is measured at a low concentration to a high concentration. It can be seen that the measurement range has expanded.
That is, it was confirmed that the measurement method and the measurement reagent can accurately measure CRP in a wide range of concentrations from low concentration to high concentration.

(Method for measuring CRP in sample of the present invention and confirmation of effect of measurement reagent-2)
The present invention comprising latex particles immobilized with an antibody (antibody-2) that binds to CRP in a calcium ion-dependent manner and latex particles immobilized with an antibody (antibody-4) that binds to a CRP in a calcium ion-independent manner. The CRP in the sample was measured using the CRP measurement reagent, and the effect was confirmed.
1. Reagent (1) 1st reagent 100% tris (hydroxymethyl) aminomethane containing 1% (w / v) BSA, 2M sodium chloride, 0.2mM calcium chloride dihydrate and 0.05% sodium azide -A hydrochloric acid buffer (pH 8.0 (20 ° C)) was prepared and used as the first reagent.
(2) Preparation of Second Reagent (a) Antibody-2 Immobilized Latex Particle Suspension To 0.094 mL of a 10% suspension of latex particles having an average particle size of 0.1 μm, (2) of Example 1-2 Antibody-2 [specific binding substance (antibody) that binds to CRP in a calcium ion-dependent manner) mixed with 6.7 mM MES buffer (pH 5.0 (20 ° C.)) at a concentration of 0.1 g / dL 0.5098 mL was added and it stirred at 5 degreeC overnight.
Next, after removing the supernatant by centrifugation, 100 mM glycine buffer solution (pH 9.5 (20 ° C.) containing 1.0% BSA) is added to the precipitate and suspended, and the mixture is stirred at room temperature for 30 minutes to block. Processed.
Next, the precipitate was recovered by centrifugation, and then suspended in a 0.05% aqueous sodium azide solution so that the absorbance at a wavelength of 585 nm was 9.5 OD.
This was diluted with a 0.05% aqueous sodium azide solution to prepare a 0.120% suspension of latex particles on which antibody-2 (an antibody that binds to CRP in a calcium ion-dependent manner) was immobilized.
This was designated as antibody-2 immobilized latex particle suspension.
(B) Preparation of antibody-4-immobilized latex particle suspension As described in (b) of (2) of Example 3 above, antibody-4 (binding to CRP independent of calcium ions) 0.036% suspension of latex particles on which antibody was immobilized) was prepared, and this was designated as antibody-4 immobilized latex particle suspension.
(C) Preparation of Second Reagent [Antibody-2] 5 mL of the antibody-2 immobilized latex particle suspension prepared in (a) above and 5 mL of 0.05% sodium azide aqueous solution were mixed. A suspension containing 0.060% “latex particles immobilized with antibody-2 (an antibody that binds to CRP in a calcium ion-dependent manner)” was prepared.
This was designated as a second reagent [antibody-2].
(D) Preparation of second reagent [antibody-4] 5 mL of the antibody-4-immobilized latex particle suspension prepared in (b) above and 5 mL of 0.05% aqueous sodium azide solution were mixed. A suspension containing 0.018% of “antibody-4 (an antibody that binds to CRP in a calcium ion-independent manner)” was prepared.
This was designated as a second reagent [antibody-4].
(E) Preparation of second reagent [antibody-2 / antibody-4] 5 mL of antibody-2 immobilized latex particle suspension prepared in (a) above and antibody-4 immobilization prepared in (b) above 5 mL of the latex particle suspension was mixed with 0.060% of “antibody-2 (an antibody that binds to CRP in a calcium ion-dependent manner)” and 0.018% of “antibody— A suspension containing 4 (latex particles immobilized with CRP in an independent manner of calcium ions) was prepared.
This was designated as a second reagent [antibody-2 / antibody-4].
2. Samples were carried out as described in 2 (1) and (2) of Example 3, and samples of the following CRP concentrations were prepared.
(A) Sample 1: 0 mg / dL
(B) Sample 2: 0.5 mg / dL
(C) Sample 3: 2.0 mg / dL
(D) Sample 4: 6.0 mg / dL
(E) Sample 5: 18.0 mg / dL
(F) Sample 6: 30.0 mg / dL
3. Measurement of CRP in a sample (1) Measurement procedure As a second reagent, the second reagent [antibody-2] of (2), (c) above, (2) the second reagent [antibody-4], and The CRP in the sample was measured as described in Example 3-3 (1) except that the second reagent [antibody-2 / antibody-4] of (e) was used. A measured value (absorbance difference) was obtained.
(2) Measurement results Table 4 and Fig. 7 show the absorbance difference obtained by measuring CRP in the sample in (1).
In FIG. 7, the horizontal axis represents the CRP concentration (mg / dL) in the sample, and the vertical axis represents the value of absorbance difference obtained by measurement [absorbance difference × 10,000] (main wavelength 570 nm, subwavelength 800 nm. ).
In FIG. 7, “Δ” indicates a measurement result (absorbance difference value) when “second reagent [antibody-2]” is used as the second reagent, and “◯” indicates “second reagent”. The measurement results (absorbance difference values) when using the second reagent [antibody-4] are shown, and “■” uses “second reagent [antibody-2 / antibody-4]” as the second reagent. The measurement results (absorbance difference values) are shown.

4). Consideration (a) From Table 4 and FIG. 7, the anti-CRP antibody of the “carrier (latex particle) on which the anti-CRP antibody is immobilized” to be contained in the CRP measurement reagent in the sample is expressed as “calcium ion-dependent CRP. When the above-mentioned “antibody-2” which is a “specific binding substance (antibody) to be bound” is used (when the second reagent is “second reagent [antibody-2]”), It can be seen that when CRP is in the low concentration range, the sensitivity is low, but the sensitivity is high in the medium concentration range to the high concentration range and has the quantitativeness of measurement.
(B) From Table 4 and FIG. 7, the “antibody-4”, which is a “specific binding substance (antibody) that binds to CRP independent of calcium ions”, is used as the anti-CRP antibody. (If the second reagent is “second reagent [antibody-4]”), the CRP in the sample is highly sensitive when in the low concentration range, but the medium concentration range to the high concentration range. It can be seen that the measurement does not have the quantitativeness of measurement.
(C) On the other hand, from Table 4 and FIG. 7, the anti-CRP antibody of the “carrier (latex particles) on which the anti-CRP antibody is immobilized” to be contained in the CRP measurement reagent in the sample is “calcium ion-dependent” The above-mentioned “antibody-2” which is a specific binding substance (antibody) that binds to CRP ”and the“ antibody ”which is a“ specific binding substance (antibody) that binds to CRP independently of calcium ions ” -4 ”(when the second reagent is“ second reagent [antibody-2 / antibody-4] ”), the CRP in the sample is measured at a low concentration to a high concentration. It can be seen that the measurement range has expanded.
That is, it was confirmed that the measurement method and the measurement reagent can accurately measure CRP in a wide range of concentrations from low concentration to high concentration.

(Method for measuring CRP in sample of the present invention and confirmation of effect of measurement reagent-3)
Latex particles immobilized with antibody (antibody-3) that binds to CRP in a calcium ion-dependent manner, and latex particles immobilized with antibody (antibody-4) that binds to CRP in a calcium ion-independent manner The CRP in the sample was measured using the CRP measurement reagent, and the effect was confirmed.
1. Reagent (1) 1st reagent 100% tris (hydroxymethyl) aminomethane containing 1% (w / v) BSA, 2M sodium chloride, 0.2mM calcium chloride dihydrate and 0.05% sodium azide -A hydrochloric acid buffer (pH 8.0 (20 ° C)) was prepared and used as the first reagent.
(2) Second reagent (a) Preparation of antibody-3 immobilized latex particle suspension To 0.094 mL of 10% suspension of latex particles having an average particle size of 0.1 μm, (2) of Example 1-2. Antibody-3 [specific binding substance that binds to CRP in a calcium ion-dependent manner (antibody)] mixed with 6.7 mM MES buffer (pH 5.0 (20 ° C.)) at a concentration of 0.1 g / dL 0.5098 mL was added and it stirred at 5 degreeC overnight.
Next, after removing the supernatant by centrifugation, 100 mM glycine buffer solution (pH 9.5 (20 ° C.) containing 1.0% BSA) is added to the precipitate and suspended, and the mixture is stirred at room temperature for 30 minutes to block. Processed.
Next, the precipitate was recovered by centrifugation, and then suspended in a 0.05% aqueous sodium azide solution so that the absorbance at a wavelength of 585 nm was 9.5 OD.
This was diluted with a 0.05% aqueous sodium azide solution to prepare a 0.120% suspension of latex particles on which antibody-3 (an antibody that binds to CRP in a calcium ion-dependent manner) was immobilized.
This was designated as an antibody-3 immobilized latex particle suspension.
(B) Preparation of antibody-4-immobilized latex particle suspension As described in (b) of (2) of Example 3 above, antibody-4 (binding to CRP independent of calcium ions) 0.036% suspension of latex particles on which antibody was immobilized) was prepared, and this was designated as antibody-4 immobilized latex particle suspension.
(C) Preparation of Second Reagent [Antibody-3] 5 mL of the antibody-3 immobilized latex particle suspension prepared in (a) above was mixed with 5 mL of 0.05% sodium azide aqueous solution. A suspension containing 0.060% “latex particles immobilized with antibody-3 (an antibody that binds to CRP in a calcium ion-dependent manner)” was prepared.
This was designated as a second reagent [antibody-3].
(D) Preparation of second reagent [antibody-4] 5 mL of the antibody-4-immobilized latex particle suspension prepared in (b) above and 5 mL of 0.05% aqueous sodium azide solution were mixed. A suspension containing 0.018% of “antibody-4 (an antibody that binds to CRP in a calcium ion-independent manner)” was prepared.
This was designated as a second reagent [antibody-4].
(E) Preparation of second reagent [antibody-3 / antibody-4] 5 mL of antibody-3 immobilized latex particle suspension prepared in (a) above and antibody-4 immobilization prepared in (b) above 5 mL of the latex particle suspension was mixed with 0.060% of “antibody-3 (an antibody that binds to CRP in a calcium ion-dependent manner)” and 0.018% of “antibody— A suspension containing 4 (latex particles immobilized with CRP in an independent manner of calcium ions) was prepared.
This was designated as a second reagent [antibody-3 / antibody-4].
2. Samples were carried out as described in 2 (1) and (2) of Example 3, and samples of the following CRP concentrations were prepared.
(A) Sample 1: 0 mg / dL
(B) Sample 2: 0.5 mg / dL
(C) Sample 3: 2.0 mg / dL
(D) Sample 4: 6.0 mg / dL
(E) Sample 5: 18.0 mg / dL
(F) Sample 6: 30.0 mg / dL
3. Measurement of CRP in a sample (1) Measurement procedure As a second reagent, the second reagent [antibody-3] of (2), (c) of (1), the second reagent [antibody-4] of (d), and The CRP in the sample was measured as described in 3 (1) of Example 3 except that the second reagent [antibody-3 / antibody-4] of (e) was used. A measured value (absorbance difference) was obtained.
(2) Measurement results Table 5 and Fig. 8 show the absorbance difference obtained by measuring CRP in the sample in (1).
In FIG. 8, the abscissa indicates the CRP concentration (mg / dL) in the sample, and the ordinate indicates the value of absorbance difference obtained by measurement [absorbance difference × 10,000] (main wavelength 570 nm, subwavelength 800 nm. ).
In FIG. 8, “Δ” indicates a measurement result (absorbance difference value) when “second reagent [antibody-3]” is used as the second reagent, and “◯” indicates “second reagent”. The measurement results (absorbance difference values) when using the second reagent [antibody-4] are shown, and “■” used “second reagent [antibody-3 / antibody-4]” as the second reagent. The measurement results (absorbance difference values) are shown.

4). Discussion (a) From Table 5 and FIG. 8, the anti-CRP antibody of “carrier (latex particles) on which anti-CRP antibody is immobilized” to be contained in the CRP measurement reagent in the sample is expressed as “calcium ion-dependent CRP. When the above-mentioned “antibody-3” which is a “specific binding substance (antibody) to be bound” is used (when the second reagent is “second reagent [antibody-3]”), It can be seen that when CRP is in the low concentration range, the sensitivity is low, but the sensitivity is high in the medium concentration range to the high concentration range and has the quantitativeness of measurement.
(B) From Table 5 and FIG. 8, the above-mentioned “antibody-4”, which is a “specific binding substance (antibody) that binds to CRP independent of calcium ions”, is used as the anti-CRP antibody. If the second reagent is “second reagent [antibody-4]”, the CRP in the sample is highly sensitive when it is in the low concentration range, but the medium concentration range to the high concentration range. It can be seen that the measurement does not have quantitative measurement.
(C) On the other hand, from Table 5 and FIG. 8, the anti-CRP antibody of “carrier (latex particles) immobilized with anti-CRP antibody” contained in the CRP measurement reagent in the sample is “calcium ion-dependent”. The above-mentioned “antibody-3” which is a specific binding substance (antibody) that binds to CRP ”and the“ antibody ”which is a“ specific binding substance (antibody) that binds to CRP independently of calcium ions ” -4 ”(when the second reagent is“ second reagent [antibody-3 / antibody-4] ”), the CRP in the sample is measured at a low concentration to a high concentration. It can be seen that the measurement range has expanded.
That is, it was confirmed that the measurement method and the measurement reagent can accurately measure CRP in a wide range of concentrations from low concentration to high concentration.

(Confirmation of anti-CRP antibody-2)
An anti-CRP antibody that binds to CRP in a calcium ion-dependent manner by a sandwich method using an ELISA method (enzyme immunoassay) using a peroxidase-labeled antibody and a CRP-immobilized plate, or is independent of calcium ion It was determined whether the antibody was binding to CRP.
1. Preparation of Reagent (1) Preparation of peroxidase-labeled antibody A peroxidase-labeled antibody was prepared as described in Example 1, 1 (1).
(2) Preparation of Cleaning Solution A cleaning solution was prepared as described in 1 (2) of Example 1 above.
(3) Preparation of CRP-immobilized plate A CRP-immobilized plate was prepared as described in 1 (3) of Example 1 above.
(4) Preparation of Coloring Solution A coloring solution was prepared as described in Example 1, 1 (4).
2. Preparation of Antibody Sample (1) Preparation of Diluent A Diluent A was prepared as described in Example 1, 2 (1).
(2) Preparation of Diluent B Diluent B was prepared as described in 2 (2) of Example 1.
(3) Anti-CRP antibody The following commercially available antibody was prepared as an anti-CRP antibody.
Antibody-5: Mouse anti-human CRP monoclonal antibody [clone: CRB-028] (Japan Biotest Laboratories)
(4) Preparation of antibody sample (a) Calcium ion-containing anti-CRP antibody sample Antibody-5 (anti-CRP antibody) of (3) above is diluted with the diluent A prepared in (1) above, and anti-CRP antibody A calcium ion-containing anti-CRP antibody sample having a concentration of 0.010 μg / mL, 0.10 μg / mL, 1.0 μg / mL, 10.0 μg / mL, or 100.0 μg / mL was prepared.
Further, the diluent A prepared in the above (1) was used as a calcium ion-containing anti-CRP antibody sample having an anti-CRP antibody concentration of 0 μg / mL.
(B) EDTA-containing / anti-CRP antibody sample Antibody-5 (anti-CRP antibody) of (3) above is diluted with diluent B prepared in (2) above, and the concentration of anti-CRP antibody is 0.010 μg / mL EDTA-containing anti-CRP antibody samples of 0.10 μg / mL, 1.0 μg / mL, 10.0 μg / mL, or 100.0 μg / mL were prepared.
Further, the diluent B prepared in the above (2) was used as an EDTA-containing anti-CRP antibody sample having an anti-CRP antibody concentration of 0 μg / mL.
3. Measurement by ELISA method (1) Measurement (b) Washing was performed by adding 400 μL of the washing solution prepared in (2) above to each well of the CRP-immobilized plate prepared in (1) above. This washing was performed 5 times in total.
(B) Thereafter, for antibody-5 (anti-CRP antibody) prepared in (2) of (4) above, the concentration of the anti-CRP antibody was 0 μg / mL, 0.010 μg / mL, 0.10 μg / mL. , 1.0 μg / mL, 10.0 μg / mL, or 100.0 μg / mL of calcium ion-containing anti-CRP antibody sample (1 type of anti-CRP antibody × 6 concentration = 6 types in total) Antigen-antibody reaction of CRP immobilized on each well of CRP-immobilized plate and added to each well of CRP-immobilized plate washed in a) and left at 37 ° C. for 2 hours. I was allowed to do.
At this time, the CRP immobilized in each well of the CRP-immobilized plate becomes calcium-binding CRP by adding and contacting the calcium ion-containing / anti-CRP antibody sample.
Therefore, at this time, the binding property of the anti-CRP antibody to the calcium-binding CRP immobilized in each well is tested.
(C) Thereafter, the calcium ion-containing anti-CRP antibody sample in each well of the CRP-immobilized plate is removed by aspiration, and the washing solution prepared in (1) above is added to each well in an amount of 400 μL per well. This was washed a total of 5 times.
(D) Next, 50 μL of the peroxidase-labeled antibody prepared in (1) above was added to each well of the CRP-immobilized plate and reacted at 37 ° C. for 1 hour.
(E) Next, each well of the CRP-immobilized plate was washed by adding 400 μL of the washing solution prepared in (1) above per well, and this washing was performed 5 times in total.
(F) Thereafter, 100 μL of the color developing solution prepared in (1) above was added to each well of the CRP-immobilized plate and allowed to react at room temperature for 30 minutes.
(G) Thereafter, the absorbance at 415 nm of the reaction solution in each well of the CRP-immobilized plate was measured using a microplate reader (BioRad; Model 3550).
(H) Anti-CRP antibody containing calcium ion having an anti-CRP antibody concentration of Antibody-10 of 0.010 μg / mL, 0.10 μg / mL, 1.0 μg / mL, 10.0 μg / mL, or 100.0 μg / mL From the measured value (absorbance) of the sample, the measured value (absorbance) of the calcium ion-containing anti-CRP antibody sample having an anti-CRP antibody concentration of 0 μg / mL was subtracted to determine the value of the absorbance difference.
(I) In place of (b), in place of the calcium ion-containing anti-CRP antibody sample (one type of anti-CRP antibody x 6 concentration = 6 types in total), an EDTA-containing anti-CRP antibody sample [one type of anti-CRP antibody x 6 Except for adding 50 μL of the concentration = 6 types in total) to each well of the CRP-immobilized plate, the operation was performed as described in the above (a) to (h), and the absorbance difference value was obtained. .
In this measurement of the EDTA-containing / anti-CRP antibody sample, the calcium ions bound to the CRP immobilized in each well of the CRP-immobilized plate by the addition and contact of the EDTA-containing / anti-CRP antibody sample were: The CRP immobilized in each of the wells by binding with EDTA · 2Na is a calcium non-binding CRP.
Therefore, at this time, the binding property of the anti-CRP antibody to the calcium non-binding CRP immobilized in each well is tested.
(2) Measurement results In the above (1), calcium ion-containing anti-CRP antibody sample (one type of anti-CRP antibody × 6 concentration = 6 types in total) and EDTA-containing anti-CRP antibody sample [one type of anti-CRP antibody × 6 Concentration = total 6 types), and the absorbance difference values obtained in the test of the binding of anti-CRP antibody (antibody-5) to calcium-bound CRP and calcium-unbound CRP are shown in Table 6 and FIG.

In FIG. 9, the horizontal axis represents the concentration of anti-CRP antibody (μg / mL) in the antibody sample added to the well of the CRP-immobilized plate, and the vertical axis represents the absorbance difference value (415 nm) obtained by the measurement. Represents.
In FIG. 9, “●” indicates the measurement result (absorbance difference) when each calcium ion-containing / anti-CRP antibody sample is measured, and “▲” indicates each EDTA-containing / anti-CRP antibody sample. The measurement results (absorbance difference) are shown.
4). Discussion From the above examination results, in antibody-5, the curve of the measurement result (calcium ion-containing anti-CRP antibody sample) showing the binding property of the anti-CRP antibody to the calcium-binding CRP, and the anti-CRP antibody not binding to calcium It can be seen that the curves of the measurement results (EDTA-containing anti-CRP antibody sample) showing the binding property to the type CRP are hardly separated.
That is, it can be seen that the antibody-5 binds to almost the same extent as both calcium-bound CRP and calcium-unbound CRP.
From this, it can be determined that the antibody-5 is a “specific binding substance (antibody) that binds to CRP independent of calcium ions”.

(Confirmation of anti-CRP antibody-immobilized latex particles-2)
The latex particles immobilized with the antibody-5, which is an antibody that binds to CRP in a calcium ion-independent manner, were confirmed for reactivity with CRP in the sample.
1. Measurement Reagent (1) First Reagent As described in Example 1, (1) (1), a first reagent was prepared.
(2) Second reagent / second reagent [antibody-5]
The antibody-1 in (2) (b) in Example 2 (1) is changed to the antibody-5 in Example (2) (3) -5 [specific binding substance (antibody) that binds to CRP in a calcium ion-independent manner. )] Except that the procedure is as described in Example 2 (1) (2) (A) to prepare a 0.060% suspension of latex particles immobilized with antibody-5. did.
This was designated as a second reagent [antibody-5].
2. Sample (1) Preparation of Sample Diluent A sample diluent was prepared as described in 2 (1) of Example 2.
(2) Preparation of Samples As described in 2 (2) of Example 2, the following samples were prepared.
(A) Sample 1: 0 mg / dL
(B) Sample 2: 0.5 mg / dL
(C) Sample 3: 2.0 mg / dL
(D) Sample 4: 6.0 mg / dL
(E) Sample 5: 18.0 mg / dL
(F) Sample 6: 30.0 mg / dL
3. Measurement of CRP in sample (1) Measurement procedure (A) Measurement was performed using a Hitachi-7180 type automatic analyzer (manufactured by Hitachi, Ltd.).
First, 3 μL of Sample 1, Sample 2, Sample 3, Sample 4, Sample 5, or Sample 6 of (2) above was added to a measurement cell (cuvette).
Next, 100 μL of the first reagent (1) was added to these measurement cells (cuvettes) and mixed.
These measurement cells (cuvettes) were allowed to stand at 37 ° C.
(B) At 4 minutes and 34 seconds (16th point) after the addition of the first reagent, the liquid mixture in the measurement cell (cuvette) is further added to the second reagent of the above (2) [ 100 μL of Antibody-5] was added and mixed.
(C) At 5 minutes 09 seconds (18th point) after the addition of the first reagent, the absorbance of the liquid mixture (main wavelength 570 nm, subwavelength 800 nm) in these measurement cells (cuvette) was blinded to the sample. As measured.
These measurement cells (cuvettes) were allowed to stand at 37 ° C. for reaction.
As a result, an antigen-antibody reaction between the anti-CRP antibody immobilized on the latex particles and the CRP contained in the sample was performed to generate latex particle aggregates.
(D) At 9 minutes and 54 seconds (34 points) after the addition of the first reagent, the absorbance (main wavelength 570 nm, subwavelength 800 nm) of the reaction mixture in this measurement cell (cuvette) It was measured as a measured value.
(E) The absorbance (sample blind) measured in (b) was subtracted from the absorbance (measured value) measured in (d) above to obtain an absorbance difference.
This difference in absorbance is proportional to the amount (concentration) of CRP contained in the sample.
(2) Measurement results Table 7 and FIG. 10 show the absorbance difference obtained by measuring CRP in the sample in (1).
In FIG. 10, the horizontal axis represents the CRP concentration (mg / dL) in the sample, and the vertical axis represents the value of absorbance difference obtained by measurement [absorbance difference × 10,000] (main wavelength 570 nm, subwavelength 800 nm. ).
In FIG. 10, “▲” indicates a measurement result (absorbance difference value) when “second reagent [antibody-5]” is used as the second reagent.

4). Discussion From Table 7 and FIG. 10, “anti-CRP antibody binding to CRP is independent of calcium ion” as an anti-CRP antibody of “carrier (latex particle) on which anti-CRP antibody is immobilized” contained in a CRP measurement reagent in a sample. When the above-mentioned “antibody-5” which is a “specific binding substance (antibody)” is used, when the CRP in the sample is in a low concentration range, the reactivity with the CRP is high and the measurement sensitivity is high. From the medium concentration range to the high concentration range, even if the CRP concentration in the sample increases, the value of the absorbance difference does not increase or decreases, that is, even if the CRP concentration in the sample increases, It can be seen that the amount produced does not increase or decreases.
Therefore, when the above-mentioned “antibody-5” is used, that is, when “specific binding substance (antibody) that binds to CRP independent of calcium ion” is used, it is highly sensitive in a low concentration range. In addition, it was confirmed that the measurement was not quantitative in the middle concentration range to the high concentration range.

(Method for measuring CRP in the sample of the present invention and confirmation of the effect of the measuring reagent-4)
The present invention comprising latex particles immobilized with an antibody (antibody-1) that binds to CRP in a calcium ion-dependent manner and latex particles immobilized with an antibody (antibody-4) that binds to a CRP in a calcium ion-independent manner. The CRP in the sample was measured using the CRP measurement reagent, and the effect was confirmed.
1. Measurement reagent (1) 1st reagent The 1st reagent was prepared as the description of 1 of said Example 3 (1).
(2) Preparation of Second Reagent (a) Antibody-1 Immobilized Latex Particle Suspension As described in Example 3, 1 (2) (a), antibody-1 immobilized latex particle suspension A liquid was prepared.
(B) Preparation of Antibody-4 Immobilized Latex Particle Suspension An antibody-4 immobilized latex particle suspension was prepared as described in (2) of (2) in Example 3 above.
(C) Preparation of second reagent [antibody-1] A second reagent [antibody-1] was prepared as described in (c) of (2) of Example 3-1.
(D) Preparation of Second Reagent [Antibody-4] As described in Example 2, 1 (2) (d), a second reagent [Antibody-4] was prepared.
(E) Preparation of Second Reagent [Antibody-1 / Antibody-4] As described in (2) (e) of 1 of Example 3, the second reagent [antibody-1 / antibody-4] was prepared. Prepared.
2. Samples were performed as described in 2 (1) and (2) of Example 3 to prepare samples of the following CRP concentrations.
(A) Sample 1: 0 mg / dL
(B) Sample 2: 0.5 mg / dL
(C) Sample 3: 2.0 mg / dL
(D) Sample 4: 6.0 mg / dL
(E) Sample 5: 18.0 mg / dL
(F) Sample 6: 30.0 mg / dL
3. Measurement of CRP in sample (1) Measurement procedure As the second reagent, the second reagent [antibody-1] of (2), (c) of (1), the second reagent [antibody-4] of (d), and Using the second reagent [antibody-1 / antibody-4] of (e), CRP in the sample was measured as described in 3 (1) of Example 3, and the measured value ( Absorbance difference) was obtained.
(2) Measurement results Table 8 and FIG. 11 show the absorbance difference obtained by measuring CRP in the sample in (1).
In FIG. 11, the abscissa indicates the CRP concentration (mg / dL) in the sample, and the ordinate indicates the value of absorbance difference obtained by measurement [absorbance difference × 10,000] (main wavelength 570 nm, subwavelength 800 nm. ).
In FIG. 11, “Δ” indicates a measurement result (absorbance difference value) when “second reagent [antibody-1]” is used as the second reagent, and “◯” indicates “second reagent”. The measurement results (absorbance difference values) when using the second reagent [antibody-4] are shown, and “■” uses “second reagent [antibody-1 / antibody-4]” as the second reagent. The measurement results (absorbance difference values) are shown.

4). Discussion (a) As can be seen from the experimental results of Example 2, the “calcium ion” is used as an anti-CRP antibody of a “carrier (latex particle) on which an anti-CRP antibody is immobilized” to be contained in a CRP measurement reagent in a sample. When the above-mentioned “antibody-1”, which is a specific binding substance (antibody) that binds to CRP in a dependent manner, is used (when the second reagent is “second reagent [antibody-1]”) ) Although the sensitivity is low when the CRP in the sample is in the low concentration range, the sensitivity is high in the medium concentration range to the high concentration range and has a quantitative property of measurement.
(B) As can be seen from the experimental results of Example 2, the above-mentioned “antibody” which is a “specific binding substance (antibody) that binds to CRP independent of calcium ions” as the anti-CRP antibody. -4 "(when the second reagent is" second reagent [antibody-4] "), the CRP in the sample is highly sensitive when it is in the low concentration range, but the medium concentration The measurement is not quantitative in the range from high to high.
(C) On the other hand, as an anti-CRP antibody of “carrier (latex particles) on which anti-CRP antibody is immobilized” to be contained in a CRP measurement reagent in a sample, “specific binding to CRP in a calcium ion-dependent manner” The above-mentioned “antibody-1” which is a “binding substance (antibody)” and the above “antibody-4” which is a “specific binding substance (antibody) that binds to CRP in a calcium ion-independent manner” are used in combination. (If the second reagent is “second reagent [antibody-1 / antibody-4]”), the measurement range should expand from a low concentration to a high concentration in the measurement of CRP in the sample. I understand.
That is, it was confirmed that the measurement method and the measurement reagent can accurately measure CRP in a wide range of concentrations from low concentration to high concentration.

(Confirmation of effect of measuring method and measuring reagent of CRP in sample of present invention-5)
The present invention comprising latex particles immobilized with an antibody (antibody-2) that binds to CRP in a calcium ion-dependent manner and latex particles immobilized with an antibody (antibody-4) that binds to a CRP in a calcium ion-independent manner. The CRP in the sample was measured using the CRP measurement reagent, and the effect was confirmed.
1. Measurement Reagent (1) First Reagent As described in Example 4, 1 (1), a first reagent was prepared.
(2) Preparation of Second Reagent (a) Antibody-2 Immobilized Latex Particle Suspension As described in Example 2, 1 (2) (a), antibody-2 immobilized latex particle suspension A liquid was prepared.
(B) Preparation of Antibody-4 Immobilized Latex Particle Suspension Antibody-4 immobilized latex particle suspension was prepared as described in (2) of (2) of Example 4 above.
(C) Preparation of Second Reagent [Antibody-2] The second reagent [Antibody-2] was prepared as described in Example 4, 1 (2) (c).
(D) Preparation of Second Reagent [Antibody-4] As described in Example 2, 1 (2) (d), a second reagent [Antibody-4] was prepared.
(E) Preparation of Second Reagent [Antibody-2 / Antibody-4] As described in (2) (e) in Example 4-1, the second reagent [antibody-2 / antibody-4] was prepared. Prepared.
2. Samples were performed as described in 2 (1) and (2) of Example 3 to prepare samples of the following CRP concentrations.
(A) Sample 1: 0 mg / dL
(B) Sample 2: 0.5 mg / dL
(C) Sample 3: 2.0 mg / dL
(D) Sample 4: 6.0 mg / dL
(E) Sample 5: 18.0 mg / dL
(F) Sample 6: 30.0 mg / dL
3. Measurement of CRP in a sample (1) Measurement procedure As a second reagent, the second reagent [antibody-2] of (2), (c) above, (2) the second reagent [antibody-4], and Using the second reagent [antibody-2 / antibody-4] of (e), CRP in the sample was measured as described in 3 (1) of Example 3, and the measured value ( Absorbance difference) was obtained.
(2) Measurement results Table 9 and Fig. 12 show the absorbance difference obtained by measuring CRP in the sample in (1).
In FIG. 12, the horizontal axis represents the CRP concentration (mg / dL) in the sample, and the vertical axis represents the absorbance difference value obtained by measurement [absorbance difference × 10,000] (main wavelength 570 nm, subwavelength 800 nm. ).
In FIG. 12, “Δ” indicates a measurement result (absorbance difference value) when “second reagent [antibody-2]” is used as the second reagent, and “◯” indicates “second reagent”. The measurement results (absorbance difference values) when using the second reagent [antibody-4] are shown, and “■” uses “second reagent [antibody-2 / antibody-4]” as the second reagent. The measurement results (absorbance difference values) are shown.
4). Discussion (a) As can be seen from the experimental results of Example 2, the “calcium ion” is used as an anti-CRP antibody of a “carrier (latex particle) on which an anti-CRP antibody is immobilized” to be contained in a CRP measurement reagent in a sample. When the above-mentioned “antibody-2”, which is a specific binding substance (antibody) that binds to CRP in a dependent manner, is used (when the second reagent is “second reagent [antibody-2]”) ) Although the sensitivity is low when the CRP in the sample is in the low concentration range, the sensitivity is high in the medium concentration range to the high concentration range and has a quantitative property of measurement.
(B) As can be seen from the experimental results of Example 2, the above-mentioned “antibody” which is a “specific binding substance (antibody) that binds to CRP independent of calcium ions” as the anti-CRP antibody. -4 "(when the second reagent is" second reagent [antibody-4] "), the CRP in the sample is highly sensitive when it is in the low concentration range, but the medium concentration The measurement is not quantitative in the range from high to high.
(C) On the other hand, as an anti-CRP antibody of “carrier (latex particles) on which anti-CRP antibody is immobilized” to be contained in a CRP measurement reagent in a sample, “specific binding to CRP in a calcium ion-dependent manner” The above-mentioned “antibody-2” which is a “binding substance (antibody)” and the above “antibody-4” which is a “specific binding substance (antibody) that binds to CRP in a calcium ion-independent manner” are used in combination. (If the second reagent is “second reagent [antibody-2 / antibody-4]”), the measurement range should expand from a low concentration to a high concentration in the measurement of CRP in the sample. I understand.
That is, it was confirmed that the measurement method and the measurement reagent can accurately measure CRP in a wide range of concentrations from low concentration to high concentration.

(Confirmation of effect of measuring method and measuring reagent of CRP in sample of present invention-6)
Latex particles immobilized with antibody (antibody-3) that binds to CRP in a calcium ion-dependent manner, and latex particles immobilized with antibody (antibody-4) that binds to CRP in a calcium ion-independent manner The CRP in the sample was measured using the CRP measurement reagent, and the effect was confirmed.
1. Measurement reagent (1) 1st reagent The 1st reagent was prepared as the description of 1 of said Example 5 (1).
(2) Second reagent (a) Preparation of antibody-3 immobilized latex particle suspension Antibody-3 immobilized latex particle suspension as described in (2) (b) of Example 5-1 A liquid was prepared.
(B) Preparation of Antibody-4 Immobilized Latex Particle Suspension An antibody-4 immobilized latex particle suspension was prepared as described in (2) of (2) of Example 5 above.
(C) Preparation of Second Reagent [Antibody-3] A second reagent [Antibody-3] was prepared as described in Example 5, 1 (2) (c).
(D) Preparation of Second Reagent [Antibody-4] As described in Example 5, 1 (2) (d), a second reagent [Antibody-4] was prepared.
(E) Preparation of second reagent [antibody-3 / antibody-4] As described in (5) of (2) in Example 5-1 above, the second reagent [antibody-3 / antibody-4] was prepared. Prepared.
2. Samples were performed as described in 2 (1) and (2) of Example 3 to prepare samples of the following CRP concentrations.
(A) Sample 1: 0 mg / dL
(B) Sample 2: 0.5 mg / dL
(C) Sample 3: 2.0 mg / dL
(D) Sample 4: 6.0 mg / dL
(E) Sample 5: 18.0 mg / dL
(F) Sample 6: 30.0 mg / dL
3. Measurement of CRP in a sample (1) Measurement procedure As a second reagent, the second reagent [antibody-3] of (2), (c) of (1), the second reagent [antibody-4] of (d), and Using the second reagent [antibody-3 / antibody-4] of (e), the CRP in the sample was measured as described in 3 (1) of Example 3, and the measured value ( Absorbance difference) was obtained.
(2) Measurement results Table 10 and Fig. 13 show the absorbance difference obtained by measuring CRP in the sample in (1).
In FIG. 13, the horizontal axis represents the CRP concentration (mg / dL) in the sample, and the vertical axis represents the value of the absorbance difference obtained by measurement [absorbance difference × 10,000] (main wavelength 570 nm, subwavelength 800 nm. ).
In FIG. 13, “Δ” indicates a measurement result (absorbance difference value) when “second reagent [antibody-3]” is used as the second reagent, and “◯” indicates “second reagent”. The measurement results (absorbance difference values) when using the second reagent [antibody-4] are shown, and “■” used “second reagent [antibody-3 / antibody-4]” as the second reagent. The measurement results (absorbance difference values) are shown.

4). Consideration (a) As can be seen from the experimental results of Example 2, “calcium ion” is used as an anti-CRP antibody of a “carrier (latex particle) on which an anti-CRP antibody is immobilized” to be contained in a CRP measurement reagent in a sample. In the case of using “antibody-3” which is a “specific binding substance (antibody) that binds to CRP in a dependent manner” (when the second reagent is “second reagent [antibody-3]”) ) Although the sensitivity is low when the CRP in the sample is in the low concentration range, the sensitivity is high in the medium concentration range to the high concentration range and has a quantitative property of measurement.
(B) As can be seen from the experimental results of Example 2, the above-mentioned “antibody” which is a “specific binding substance (antibody) that binds to CRP independent of calcium ions” as the anti-CRP antibody. -4 "(when the second reagent is" second reagent [antibody-4] "), the CRP in the sample is highly sensitive when it is in the low concentration range, but the medium concentration The measurement is not quantitative in the range from high to high.
(C) On the other hand, as an anti-CRP antibody of “carrier (latex particle) on which anti-CRP antibody is immobilized” to be contained in a CRP measurement reagent in a sample, “specific binding to CRP in a calcium ion-dependent manner” The above-mentioned “antibody-3” which is a “binding substance (antibody)” and the above “antibody-4” which is a “specific binding substance (antibody) that binds to CRP independently of calcium ions” are used in combination. (If the second reagent is “second reagent [antibody-3 / antibody-4]”), the measurement range should be expanded from a low concentration to a high concentration in the measurement of CRP in the sample. I understand.
That is, it was confirmed that the measurement method and the measurement reagent can accurately measure CRP in a wide range of concentrations from low concentration to high concentration.

(Confirmation of effect of measuring method and measuring reagent of CRP in sample of present invention-7)
The present invention comprising latex particles immobilized with an antibody (antibody-1) that binds to CRP in a calcium ion-dependent manner and latex particles immobilized with an antibody (antibody-5) that binds to a CRP in a calcium ion-independent manner. The CRP in the sample was measured using the CRP measurement reagent, and the effect was confirmed.
1. Measurement reagent (1) 1st reagent The 1st reagent was prepared as the description of 1 of said Example 3 (1).
(2) Preparation of Second Reagent (a) Antibody-1 Immobilized Latex Particle Suspension As described in Example 3, 1 (2) (a), antibody-1 immobilized latex particle suspension A liquid was prepared.
(B) Preparation of antibody-5-immobilized latex particle suspension In place of antibody-4, antibody-5 (antibody binding to CRP in a calcium ion-independent manner) of Example 2-2 (3) is used. Otherwise, an antibody-5-immobilized latex particle suspension was prepared as described in 1 (2) (b) of Example 3 above.
(C) Preparation of second reagent [antibody-1] A second reagent [antibody-1] was prepared as described in (c) of (2) of Example 3-1.
(D) Preparation of Second Reagent [Antibody-5] Example 3 except that the antibody-4 immobilized latex particle suspension was used instead of the antibody-4 immobilized latex particle suspension. The second reagent [Antibody-5] was prepared as described in 1) (2) (d).
(E) Preparation of Second Reagent [Antibody-1 / Antibody-5] In place of the antibody-4 immobilized latex particle suspension, the antibody-5 immobilized latex particle suspension described in (b) above was used, A second reagent [antibody-1 / antibody-5] was prepared as described in (3) in (2) of Example 3 above.
2. Samples were performed as described in 2 (1) and (2) of Example 3 to prepare samples of the following CRP concentrations.
(A) Sample 1: 0 mg / dL
(B) Sample 2: 0.5 mg / dL
(C) Sample 3: 2.0 mg / dL
(D) Sample 4: 6.0 mg / dL
(E) Sample 5: 18.0 mg / dL
(F) Sample 6: 30.0 mg / dL
3. Measurement of CRP in a sample (1) Measurement procedure As a second reagent, the second reagent [antibody-1] of (2), (c) of (1), the second reagent [antibody-5] of (d), and Using the second reagent [antibody-1 / antibody-5] of (e), CRP in the sample was measured as described in 3 (1) of Example 3, and the measured value ( Absorbance difference) was obtained.
(2) Measurement results In (1), the absorbance difference obtained by measuring CRP in the sample is shown in Table 11 and FIG.
In FIG. 14, the horizontal axis represents the CRP concentration (mg / dL) in the sample, and the vertical axis represents the value of the absorbance difference obtained by measurement [absorbance difference × 10,000] (main wavelength 570 nm, subwavelength 800 nm. ).
In FIG. 14, “Δ” indicates a measurement result (absorbance difference value) when “second reagent [antibody-1]” is used as the second reagent, and “◯” indicates “second reagent”. The measurement results (absorbance difference values) when using the second reagent [antibody-5] are shown, and “■” uses “second reagent [antibody-1 / antibody-5]” as the second reagent. The measurement results (absorbance difference values) are shown.

4). Discussion (a) As can be seen from the experimental results of Example 2, the “calcium ion” is used as an anti-CRP antibody of a “carrier (latex particle) on which an anti-CRP antibody is immobilized” to be contained in a CRP measurement reagent in a sample. When the above-mentioned “antibody-1”, which is a specific binding substance (antibody) that binds to CRP in a dependent manner, is used (when the second reagent is “second reagent [antibody-1]”) ) Although the sensitivity is low when the CRP in the sample is in the low concentration range, the sensitivity is high in the medium concentration range to the high concentration range and has a quantitative property of measurement.
(B) Further, as can be seen from the experimental results of Example 7, the above-mentioned “antibody” which is a “specific binding substance (antibody) that binds to CRP in a calcium ion-independent manner” as the anti-CRP antibody. When “-5” is used (when the second reagent is “second reagent [antibody-5]”), the CRP in the sample is highly sensitive when it is in the low concentration range, but the medium concentration The measurement is not quantitative in the range from high to high.
(C) On the other hand, as an anti-CRP antibody of “carrier (latex particle) on which anti-CRP antibody is immobilized” to be contained in a CRP measurement reagent in a sample, “specific binding to CRP in a calcium ion-dependent manner” The above-mentioned “antibody-1” which is a “binding substance (antibody)” and the above “antibody-5” which is a “specific binding substance (antibody) that binds to CRP in a calcium ion-independent manner” are used in combination. (If the second reagent is “second reagent [antibody-1 / antibody-5]”), the measurement range should expand from a low concentration to a high concentration in the measurement of CRP in the sample. I understand.
That is, it was confirmed that the measurement method and the measurement reagent can accurately measure CRP in a wide range of concentrations from low concentration to high concentration.

(Method for measuring CRP in sample of the present invention and confirmation of effect of measurement reagent-8)
The present invention comprising latex particles to which an antibody that binds to CRP in a calcium ion-dependent manner (antibody-2) is immobilized, and latex particles to which an antibody that binds to CRP in a calcium ion-independent manner (antibody-5) is immobilized. The CRP in the sample was measured using the CRP measurement reagent, and the effect was confirmed.
1. Measurement reagent (1) 1st reagent The 1st reagent was prepared as the description of 1 of said Example 3 (1).
(2) Second reagent (b) Preparation of suspension of antibody-2 immobilized latex particles In place of antibody-1, antibody-2 (calcium ion-dependent) of (b) in (3) of Example 1-2 The antibody-2-immobilized latex particle suspension was prepared as described in Example 1, 1 (2) (b) except that the antibody binding to CRP was used.
(B) Preparation of antibody-5-immobilized latex particle suspension In place of antibody-4, antibody-5 (antibody binding to CRP in a calcium ion-independent manner) of Example 2-2 (3) is used. Otherwise, an antibody-5-immobilized latex particle suspension was prepared as described in 1 (2) (b) of Example 3 above.
(C) Preparation of Second Reagent [Antibody-2] Example 3 except that the antibody-1 immobilized latex particle suspension was used instead of the antibody-1 immobilized latex particle suspension. The second reagent [antibody-2] was prepared as described in 1) (2) (iii).
(D) Preparation of Second Reagent [Antibody-5] Example 3 except that the antibody-4 immobilized latex particle suspension was used instead of the antibody-4 immobilized latex particle suspension. The second reagent [Antibody-5] was prepared as described in 1) (2) (d).
(E) Preparation of Second Reagent [Antibody-2 / Antibody-5] In place of the antibody-1 immobilized latex particle suspension, the antibody-2 immobilized latex particle suspension of (a) above was used, and Except for using the antibody-5 immobilized latex particle suspension of (b) above instead of the antibody-4 immobilized latex particle suspension, As described above, a second reagent [antibody-2 / antibody-5] was prepared.
2. Samples were performed as described in 2 (1) and (2) of Example 3 to prepare samples of the following CRP concentrations.
(A) Sample 1: 0 mg / dL
(B) Sample 2: 0.5 mg / dL
(C) Sample 3: 2.0 mg / dL
(D) Sample 4: 6.0 mg / dL
(E) Sample 5: 18.0 mg / dL
(F) Sample 6: 30.0 mg / dL
3. Measurement of CRP in a sample (1) Measurement procedure As a second reagent, the second reagent [antibody-2] of (2) (c) of (1) above, the second reagent [antibody-5] of (d), and Using the second reagent [antibody-2 / antibody-5] of (e), CRP in the sample was measured as described in 3 (1) of Example 3, and the measured value ( Absorbance difference) was obtained.
(2) Measurement results Table 12 and FIG. 15 show the absorbance difference obtained by measuring CRP in the sample in (1).
In FIG. 15, the horizontal axis represents the CRP concentration (mg / dL) in the sample, and the vertical axis represents the value of the absorbance difference obtained by measurement [absorbance difference × 10,000] (main wavelength 570 nm, subwavelength 800 nm. ).
In FIG. 15, “Δ” indicates a measurement result (absorbance difference value) when “second reagent [antibody-2]” is used as the second reagent, and “◯” indicates “second reagent”. The measurement results when using the second reagent [antibody-5] are shown (absorbance difference values). “■” used “second reagent [antibody-2 / antibody-5]” as the second reagent. The measurement results (absorbance difference values) are shown.

4). Discussion (a) As can be seen from the experimental results of Example 2, the “calcium ion” is used as an anti-CRP antibody of a “carrier (latex particle) on which an anti-CRP antibody is immobilized” to be contained in a CRP measurement reagent in a sample. When the above-mentioned “antibody-2”, which is a specific binding substance (antibody) that binds to CRP in a dependent manner, is used (when the second reagent is “second reagent [antibody-2]”) ) Although the sensitivity is low when the CRP in the sample is in the low concentration range, the sensitivity is high in the medium concentration range to the high concentration range and has a quantitative property of measurement.
(B) Further, as can be seen from the experimental results of Example 7, the above-mentioned “antibody” which is a “specific binding substance (antibody) that binds to CRP in a calcium ion-independent manner” as the anti-CRP antibody. When “-5” is used (when the second reagent is “second reagent [antibody-5]”), the CRP in the sample is highly sensitive when it is in the low concentration range, but the medium concentration The measurement is not quantitative in the range from high to high.
(C) On the other hand, as an anti-CRP antibody of “carrier (latex particle) on which anti-CRP antibody is immobilized” to be contained in a CRP measurement reagent in a sample, “specific binding to CRP in a calcium ion-dependent manner” The above-mentioned “antibody-2” which is a “binding substance (antibody)” and the above “antibody-5” which is a “specific binding substance (antibody) that binds to CRP in a calcium ion-independent manner” (If the second reagent is “second reagent [antibody-2 / antibody-5]”), the measurement range should be expanded from a low concentration to a high concentration in the measurement of CRP in the sample. I understand.
That is, it was confirmed that the measurement method and the measurement reagent can accurately measure CRP in a wide range of concentrations from low concentration to high concentration.

(Method for measuring CRP in sample of the present invention and confirmation of effect of measurement reagent-9)
The present invention comprising latex particles to which an antibody that binds to CRP in a calcium ion-dependent manner (antibody-3) is immobilized, and latex particles to which an antibody that binds to CRP in a calcium ion-independent manner (antibody-5) is immobilized. The CRP in the sample was measured using the CRP measurement reagent, and the effect was confirmed.
1. Measurement reagent (1) 1st reagent The 1st reagent was prepared as the description of 1 of said Example 3 (1).
(2) Second Reagent (a) Preparation of Antibody-3 Immobilized Latex Particle Suspension In place of Antibody-1, Antibody (3) in (3), (3) in Example 1 above (dependent on calcium ions) The antibody-3-immobilized latex particle suspension was prepared as described in Example 1 (2) (b) except that the antibody binding to CRP was used.
(B) Preparation of antibody-5-immobilized latex particle suspension In place of antibody-4, antibody-5 (antibody binding to CRP in a calcium ion-independent manner) of Example 2-2 (3) is used. Otherwise, an antibody-5-immobilized latex particle suspension was prepared as described in 1 (2) (b) of Example 3 above.
(C) Preparation of second reagent [antibody-3] Example 3 except that the antibody-1 immobilized latex particle suspension was used instead of the antibody-1 immobilized latex particle suspension described in (a) above. The second reagent [Antibody-3] was prepared as described in 1) (2) (iii).
(D) Preparation of Second Reagent [Antibody-5] Example 3 except that the antibody-4 immobilized latex particle suspension was used instead of the antibody-4 immobilized latex particle suspension. The second reagent [Antibody-5] was prepared as described in 1) (2) (d).
(E) Preparation of second reagent [antibody-3 / antibody-5] In place of antibody-1 immobilized latex particle suspension, antibody-3 immobilized latex particle suspension of (a) above was used, and Except for using the antibody-5 immobilized latex particle suspension of (b) above instead of the antibody-4 immobilized latex particle suspension, As described above, a second reagent [antibody-3 / antibody-5] was prepared.
2. Samples were performed as described in 2 (1) and (2) of Example 3 to prepare samples of the following CRP concentrations.
(A) Sample 1: 0 mg / dL
(B) Sample 2: 0.5 mg / dL
(C) Sample 3: 2.0 mg / dL
(D) Sample 4: 6.0 mg / dL
(E) Sample 5: 18.0 mg / dL
(F) Sample 6: 30.0 mg / dL
3. Measurement of CRP in a sample (1) Measurement procedure As a second reagent, the second reagent [antibody-3] of (2), (c) above, (2) the second reagent [antibody-5], and Using the second reagent [antibody-3 / antibody-5] of (e), CRP in the sample was measured as described in 3 (1) of Example 3, and the measured value ( Absorbance difference) was obtained.
(2) Measurement results In (1), the absorbance difference obtained by measuring CRP in the sample is shown in Table 13 and FIG.
In FIG. 16, the horizontal axis represents the CRP concentration (mg / dL) in the sample, and the vertical axis represents the value of absorbance difference obtained by measurement [absorbance difference × 10,000] (main wavelength 570 nm, subwavelength 800 nm. ).
In FIG. 16, “Δ” indicates a measurement result (absorbance difference value) when “second reagent [antibody-3]” is used as the second reagent, and “◯” indicates “second reagent”. The measurement results (absorbance difference values) when using the second reagent [antibody-5] are shown, and “■” uses “second reagent [antibody-3 / antibody-5]” as the second reagent. The measurement results (absorbance difference values) are shown.

4). Consideration (a) As can be seen from the experimental results of Example 2, “calcium ion” is used as an anti-CRP antibody of a “carrier (latex particle) on which an anti-CRP antibody is immobilized” to be contained in a CRP measurement reagent in a sample. In the case of using “antibody-3” which is a “specific binding substance (antibody) that binds to CRP in a dependent manner” (when the second reagent is “second reagent [antibody-3]”) ) Although the sensitivity is low when the CRP in the sample is in the low concentration range, the sensitivity is high in the medium concentration range to the high concentration range and has a quantitative property of measurement.
(B) Further, as can be seen from the experimental results of Example 7, the above-mentioned “antibody” which is a “specific binding substance (antibody) that binds to CRP in a calcium ion-independent manner” as the anti-CRP antibody. When “-5” is used (when the second reagent is “second reagent [antibody-5]”), the CRP in the sample is highly sensitive when it is in the low concentration range, but the medium concentration The measurement is not quantitative in the range from high to high.
(C) On the other hand, as an anti-CRP antibody of “carrier (latex particle) on which anti-CRP antibody is immobilized” to be contained in a CRP measurement reagent in a sample, “specific binding to CRP in a calcium ion-dependent manner” The above-mentioned “antibody-3” which is a “binding substance (antibody)” and the above “antibody-5” which is a “specific binding substance (antibody) that binds to CRP in a calcium ion-independent manner” are used in combination. (If the second reagent is “second reagent [antibody-3 / antibody-5]”), the measurement range should be expanded from a low concentration to a high concentration in the measurement of CRP in the sample. I understand.
That is, it was confirmed that the measurement method and the measurement reagent can accurately measure CRP in a wide range of concentrations from low concentration to high concentration.

Claims

A specific binding substance that binds to a C-reactive protein in a calcium ion-dependent manner and a specific binding substance that binds to a C-reactive protein in a calcium ion-independent manner are each immobilized on a carrier, and the carrier-immobilized specific binding substance C-reactivity in the sample is measured by measuring a complex aggregate of the specific binding substance and the C-reactive protein produced by the specific binding reaction between the C-reactive protein contained in the sample and the C-reactive protein. A method for measuring protein.
The C-reactive protein in the sample according to claim 1, wherein the specific binding substance that binds to the C-reactive protein in a calcium ion-dependent manner is an antibody that binds to the C-reactive protein in a calcium-ion-dependent manner. How to measure.
The method for measuring C-reactive protein in a sample according to claim 1 or 2, wherein the antibody that binds to C-reactive protein in a calcium ion-dependent manner is a monoclonal antibody.
4. The specific binding substance that binds to C-reactive protein independent of calcium ions is an antibody that binds to C-reactive protein independent of calcium ions. A method for measuring C-reactive protein in the sample according to Item.
The method for measuring C-reactive protein in a sample according to any one of claims 1 to 4, wherein the antibody that binds to C-reactive protein independently of calcium ions is a monoclonal antibody.
6. The method for measuring C-reactive protein in a sample according to any one of claims 1 to 5, wherein the carrier is latex particles.
A sample comprising a specific binding substance that binds to C-reactive protein in a calcium ion-dependent manner and a carrier on which a specific binding substance that binds to C-reactive protein in a calcium ion-independent manner is immobilized A reagent for measuring C-reactive protein.
The specific binding substance that binds to the C-reactive protein in a calcium ion-dependent manner is an antibody that binds to the C-reactive protein in a calcium ion-dependent manner. The C-reactive protein in the sample according to claim 7 Measuring reagent.
The reagent for measuring C-reactive protein in a sample according to claim 7 or 8, wherein the antibody that binds to C-reactive protein in a calcium ion-dependent manner is a monoclonal antibody.
10. The specific binding substance that binds to a C-reactive protein independent of calcium ions is an antibody that binds to a C-reactive protein independent of calcium ions. A reagent for measuring C-reactive protein in the sample according to Item.
11. The reagent for measuring C-reactive protein in a sample according to any one of claims 7 to 10, wherein the antibody that binds to C-reactive protein independently of calcium ions is a monoclonal antibody.
12. The reagent for measuring C-reactive protein in a sample according to any one of claims 7 to 11, wherein the carrier is latex particles.