JP2001330614A - Solid phase to which biological activity is imparted, and method for manufacturing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a solid phase having biological activity of an antibody or the like imparted thereto and capable of achieving highly accurate immunological examination, in a short time. SOLUTION: A chemically activable part is arranged to at least a part of the surface of the solid phase, having a biologically active substance such as an antibody or the like directly bonded to the surface thereof, and the solid phase has characteristics such that the other part of the surface thereof has no interference properties with respect to the biologically active substance and shows suspension and sedimentation properties in a liquid and contains a magnetic material appropriately. A solid phase manufacturing method is also disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a solid phase to which a biologically active substance is bound. More specifically, the present invention can be effectively applied to an immunoassay carrier to which an immunological substance such as an antibody is directly bound.

[0002]

2. Description of the Related Art Conventionally, a technique using a solid phase for analyzing or extracting a biologically active substance by using the presence or absence or strength of the biological activity as an index has been widely used.

[0003] For example, an immunological test using an antibody is widely used in various blood tests such as blood type determination and platelet cross-compatibility test. For safe transfusion,
These blood tests are indispensable, and in order to prevent medical accidents, the accuracy of the test must be as high as possible and it is essential that the test can be performed quickly. In addition, it is necessary that the reagent used for the test can be stably stored for a long period of time.

At present, in order to carry out a blood test, for example, a cross-compatibility test of platelets, an operation using erythrocytes (antibody-sensitized erythrocytes) bound to an anti-IgG antibody labeled with a label that can be labeled is performed. Commonly used.

[0005] In this operation, first, a sample containing an antibody against a platelet antigen is added to a plate on which blood cells such as platelets are immobilized. Subsequently, antibody-sensitized red blood cells are added and allowed to bind to the antibody against the platelet antigen. The aggregation pattern of antibody-sensitized erythrocytes differs depending on whether or not an antibody against the platelet antigen binds to the platelet antigen. Therefore, the type of the antibody in the sample can be determined based on the pattern.

However, in this operation, since the red blood cell agglutination pattern is formed by spontaneous sedimentation of the antibody-sensitized red blood cells, it takes as long as 4 to 16 hours until a result is obtained, and it is necessary to use this method when urgency is required. Can not.

In order to shorten the operation time, it is useful to bind various bioactive substances to carriers other than erythrocytes. In general, techniques for binding various antibody bioactive substances to carriers are described below. Depending on the material of the carrier, there are an adsorption method and a covalent bonding method.

[0008] As the adsorption method, there is a method based on charge of the surface of the carrier itself, hydrophobicity of the surface, and hydrogen bonding. In the case of the adsorption method,
If the ability to adsorb proteins etc. is too strong, non-specific reactions are likely to occur, and if the reaction environment such as pH is different, the reaction amount may also change, so the reaction accuracy of biological activity is low or long-term storage is difficult Often.

In the covalent bonding method, a protein or the like is bound by chemically adding an activator or a cross-linking agent which functions as a spacer on the surface of a carrier, but the number of the added is increased. Therefore, when the amount of binding is increased, the chance of non-specific binding tends to increase accordingly. For this reason, a protection treatment is often performed so that a portion other than the target protein or the like bound thereto is covered with an organic substance (eg, albumin) that is inert to biological activity. Therefore, in the covalent bonding method, as in the case of the adsorption method, a non-specific reaction occurs and the reaction accuracy is low, or the stability of the three-dimensional structure due to denaturation or addition of the protective organic substance tends to be difficult to store for a long time. .

[0010]

SUMMARY OF THE INVENTION The present invention provides a solid phase in which a biologically active substance is directly bonded to a surface, and which can achieve a highly accurate immunoassay in a short time. The purpose is to do.

[0011]

In order to solve the above problems, the present invention provides a solid phase in which a biologically active substance is directly bonded to a surface, wherein at least a part of the surface of the solid phase is chemically bonded. A solid phase is provided which is activated and characterized in that the other part is non-interfering with said bioactive substance.

[0012]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a solid phase having a bioactive substance directly attached to a surface.

[0013] The solid phase of the present invention may typically be used for "immunological testing", ie, any testing, analysis, and testing using antibodies.

As used herein, the term "solid phase" refers to a substance in which the surface of the outer wall of particles, substrates, rods and the like and the surface of the inner wall of containers, tubes, columns, porous members and the like are at least insoluble and immobilized. Say. In addition, "biologically active substance" refers to immunological, antigen, antibody, nucleic acid, enzyme, hormone, cell, etc.
A substance having biochemical or genetic activity.

The biologically active substance is "directly bound" to the solid phase.
To mean that the group in the solid phase and the group in the biologically active substance directly form a bond. That is, in the solid phase of the present invention, no biologically active substance is bound to the solid phase via a crosslinking agent or a dye.

In order to bind the biologically active substance directly,
The solid phase of the present invention has at least a part of its surface chemically activated. Where "chemically activated"
A material having a functional group selected from the group described in the embodiment described below is changed to a state capable of binding a biologically active substance by a chemical activation treatment described in the embodiment described below. Means that

Preferably, the solid phase having the activatable material is free of interference with the biologically active substance to be immobilized.
Alternatively, it is a substance close to a living body that has almost no interference. For example, if the biological activity is antigen-antibody reactivity, the activatable material refers to a material having no or little antigenicity.

Therefore, the solid phase of the present invention is non-interfering with the biologically active substance except for the activated part.
"Non-interfering with a biologically active substance" means a charge,
It means that physical adsorption force due to adhesion or the like, affinity to biological activity, and repulsion to biological activity do not appear on the surface or are negligible. For example, when the biological activity is antigen-antibody reactive, it means that the solid phase has no or almost no antigenicity.

In order to prepare the solid phase of the present invention, an operation for chemically bonding a group in the solid phase to a group in the biologically active substance is performed.

Carriers as solid phases to which biologically active substances are to be bound include, for example, gelatin, natural rubber, polyamino acids, collagen, gum arabic, dextran, polysaccharides such as cellulose and agarose, hydrophilic polypinyl, polyethylene glycol, Acrylamide, and mixtures thereof, can be used. In order to make these materials in a solid state, a known granulation technique is applied, and 0.5 to 30 μm
Should be obtained. Also, a solid phase composed of a non-interfering material whose surface is coated with these materials may be used.

For example, the carrier can be in any form including particles, microtiter plates, microtubes.

Here, according to the study of the present inventors, gelatin and Arabic acid have been considered in that they can sufficiently utilize hydroxyl groups, and are excellent in dispersibility between particles and non-interference of the particle surface with an object to be examined or extracted. Rubber coacervate particles
(See Japanese Patent Publication No. 7-86508) was found to be one embodiment of the solid phase with the optimal combination of materials. In particular, as shown in the examples below, the coacervate particles to which the present invention is applied, the storage stability and reactivity of biological activity are extremely delicate against platelet membrane antigens,
The achievement of highly reliable performance was reported for the first time in the present invention, and is expected to be a technology that can greatly contribute to future blood transfusion tests.

As described in detail below, the groups in the solid phase include those present in the biologically active substance, such as amino, carboxyl, hydroxyl, imidazole, guanidyl, or sulfhydryl groups. Are combined. Therefore, the solid phase must contain groups that can react with these groups present in the biologically active substance to be bound to form a bond. For example, the solid phase contains free hydroxyl groups,
It preferably contains an amino group, a carboxyl group, an aldehyde group, a sulfhydryl group, or the like.

When the bioactive substance is a protein,
It must be bound to the solid phase under mild conditions in which the higher order structure can be retained. A method for immobilizing a protein on a solid phase under such conditions is itself known.

When the solid phase has an amino group or a hydroxyl group, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) or dicyclohexylcarbodiimide
(DDC) can be used to link a carboxyl group of a biologically active substance to an amino group or a hydroxyl group on a solid phase. Conversely, when the solid phase has a carboxyl group,
In a similar manner, the amino group of the biologically active substance can be bound to the carboxyl group of the solid phase.

When the solid phase has a carboxyl group, the same method as in the solid phase synthesis of peptides may be applied. More specifically, after an activated solid phase is formed by binding an activator to the solid phase and then contacted with a bioactive substance, the carboxyl group of the solid phase and the amino group of the bioactive substance are converted. It becomes possible to combine.

Here, the term "activator" refers to a substance which binds to a carboxyl group on the solid phase and activates the carboxyl group on the solid phase so that the amino group of the peptide can be bound. N-hydroxy compounds, including hydroxysuccinimide, 1-hydroxybenzotriazole, and 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine, preferably N-hydroxysuccinimide, may be used.

When activating the carboxyl group of the solid phase by using an activating agent, it is preferable to add a dehydrating agent to promote the reaction for generating the activated solid phase.

As the dehydrating agent, carbodiimide or a derivative thereof or a salt thereof, more preferably, dicyclhexylcarbodiimide (DDC), 1-ethyl-3- (3′-dimethylaminopropyl) -carbodiimide (EADC), Alternatively, diisopropylcarbodiimide (DIPC) may be used.

As described above, the solid phase of the present invention comprises a solid phase having a property such that a chemically activatable portion is disposed on at least the surface thereof and which is non-interfering with the biologically active substance. And a step of bringing a bioactive substance into contact with the activated surface of the solid phase. Such a method for producing a solid phase also falls within the scope of the present invention.

As an example, the following formula shows an operation for directly binding a biologically active substance to a solid phase using an N-hydroxy compound.

[0032]

Embedded image

When the antibody is bound to the solid phase using EADC and N-hydroxysuccinimide, the pH can be 3 to 10, but the condensing agent for condensing the moiety activated by the activating agent (in this case, Due to the effect of a dehydrating agent that dehydrates hydroxyl groups), it can be prepared only under phosphoric acid (or carbonic acid) under mild conditions near neutrality (for example, in the range of pH 5.7 to 6.6) and without hydrochloric acid. Thus, there is an advantage that handling as waste is easy. When the reaction is carried out at pH 5.7 to 8.0, most preferably around pH 6.2, the antibody easily binds to the solid phase. In this case, when the reaction is carried out in a phosphate buffer or a carbonate buffer, more preferably in a phosphate buffer, the antibody is easily bound to the solid phase.

An antibody fragment, for example, a Fab fragment may be bound instead of, or together with, the antibody to be bound to the solid phase. The antibody or fragment thereof can be any antigen,
Antibodies against each immunoglobulin class, such as human, sheep, goat, cow, horse, dog, cat, etc., are preferred.

The solid phase provided with the biological activity can preferably contain a magnetic substance. The solid phase is encapsulated, adhered,
In the case of a carrier containing a magnetic substance by coating or the like, the desired processing step can be performed in a short time by an appropriate magnetic generation means (permanent magnet, electromagnet, etc.) in each processing step such as stirring, washing, and measurement in a liquid. It can be carried out.

The solid phase of the present invention to which a bioactive substance is directly bonded has higher stability than a solid phase to which a bioactive substance is bonded via a crosslinking agent. Therefore, the solid phase to which the biologically active substance is bound via the cross-linking agent has a specific reaction in a liquid during a long-term storage, such as a change in the degree of non-specific reaction caused by the cross-linking agent or a break in the bond. Because the sex may change, it must be stored in a lyophilized state,
The carrier of the present invention is not easily affected over a long period of time even over a wide range of storage conditions, and at a low temperature of 0 to 10 ° C, more preferably at a low temperature of 0 to 4 ° C, such as an antibody bound to the carrier. It was found that the biological activity can be stably stored for a long period of time. In particular, the solid phase of the present invention to which the antibody is directly bound has higher stability than a conventional solid phase of the same type.

The solid phase of the present invention is less susceptible to nonspecific reaction than a conventional solid phase to which a biologically active substance is bound via a dye for detection. Therefore, the solid phase of the present invention
It is suitable for use in tests requiring particularly high precision, such as blood tests.

As described above, the solid phase of the present invention can be used for any test using an antibody.

Typically, the "test using an antibody" may be a test performed in the field of clinical medicine, basic science, or individual identification, preferably a clinical test.

Suitable clinical tests are blood tests such as a cross-compatibility test (blood cross-match test), screening for antibodies against cell antigens such as platelets, erythrocytes, lymphocytes, monocytes, and granulocytes, and an antibody identification test. . In particular, when using the solid phase of the present invention instead of anti-human IgG binding sheep red blood cells,
Because the test time for platelet cross-compatibility testing is significantly reduced, the solid phase of the invention is very suitable for application in platelet cross-compatibility testing. In addition, the solid phase of the present invention
The present invention can also be applied to antibody screening, antibody identification tests, cross-compatibility tests, and serotyping for antigens of pathogens such as viruses and bacteria.

The solid phase of the present invention can also be applied to other analytical methods such as immunohistochemistry and Western blotting.

For example, a cross-compatibility test of platelets using the solid phase of the present invention can be typically performed by the operation shown in FIG.

In order to carry out this operation, first, a container 2 in which platelets 1 are solid-phased or fixed on the U-shaped bottom is prepared. It is preferable that the platelet 1 is solid-phased in the container 2 using wheat germ lectin (WGA) by the method described in Japanese Patent No. 2532670. Alternatively, the platelets 1 may be fixed using glutaraldehyde or formaldehyde.

A sample containing an antibody against platelet 1, ie, anti-platelet antibody 3, usually the serum of the patient whose platelet serotype is to be determined is dispensed into container 2. In the sample,
The reaction can be terminated at 37 ° C. for 30 minutes by adding a low ionic strength solution or the like.

Subsequently, BF separation is performed to remove unbound anti-platelet antibody 3.

After dispensing the magnetic carrier 4 to which the anti-IgG antibody is directly bound into the container 2, the container 2 is placed on the magnet 5,
The magnetic carrier 4 is brought into contact with the U-shaped bottom of the container 2.

After about 2 minutes, the type of platelet antigen can be known by observing the distribution of the magnetic carrier 4.

[0048] For example, having a platelet HPA-1 ^a antigen donor, if the anti-HPA-1 ^a antibody to the patient serum is present, the surface of platelets 1 present in the U-shaped bottom portion of the container 2 anti HPA-1 ^a bioactive substance HPA-1 ^a antigen present in the binding.
Thus, thereafter, an anti-IgG antibody which is coupled directly to the magnetic carrier 4, capable of binding to anti-HPA-1 ^a antibody bound to the surface of platelets 1.

[0049] After that, if brought close to the magnet 5 in the container 2, the magnetic carrier 4, since not bound to platelets 1 via an anti-HPA-1 ^a antibody collects in the center of the U-shaped bottom portion (in FIG. 1 negative pattern). In contrast, the magnetic carrier 4 also close the magnet 5 so bound to platelets 1 via an anti-HPA-1 ^a antibody
Does not move to the center of the U-shaped bottom (positive pattern in FIG. 1).

As described above, by using the solid phase of the present invention,
It is possible to easily and accurately perform a platelet cross-matching test. Therefore, a method for determining the antigen of platelets using the above-mentioned operations and the like is also included in the scope of the present invention.

Further, since other blood tests can be performed by the same operation, a platelet antibody screening test for determining an antibody against the platelet antigen or detecting the presence or absence of the antibody, the type of erythrocyte antigen or leukocyte antigen Other blood tests are also within the scope of the invention.

Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1 This example describes a method for producing antibody-sensitized magnetic particles that can be used in a platelet cross-compatibility test. In the following embodiments including this embodiment,
OLYBIO, a commercially available antiplatelet antibody test reagent, excluding magnetic particles
Various reagents of a kit in ^TM (manufactured by Olympus Optical Co., Ltd.) and a lectin-immobilized container were used.

First, about 7.5 μm of magnetic gelatin particles (manufactured by Olympus Optical Industry Co., Ltd.) mixed with an appropriate amount of a magnetic substance using a granulation method described in JP-B-7-86508 three times with pure water. It was washed and adjusted to about 19% magnetic gelatin particles.

0.1 g of N-hydroxysuccinimide, 1-ethyl-3- (3'-dimethylaminopropyl) -carboximide
(EDAC) Add 0.1 g in order and add 0.5 g at room temperature with occasional stirring.
Allowed to react for hours.

After washing three times with pure water, 10 mL of a 200 μg / mL rabbit-derived anti-human IgG-phosphate buffer (pH 6.2) was added, and reacted at 37 ° C. for 1 hour with stirring.

After washing three times with 30 mL of PBS (pH 7.2, 4 ° C.), 0.05 w /
v% NaN ₃ -PBS (pH 7.2) (4 ° C) in 30 mL, overnight at 4 ° C (18
Hours).

After centrifugation, the antibody-sensitized magnetic gelatin particles were adjusted to 0.05 w /
v% NaN ₃ was suspended in -PBS (pH7.2).

In the following examples, it was confirmed that the magnetic gelatin particle solution thus prepared could be stably stored at 4 ° C. for 6 months or more and the reactivity did not decrease.

In the above examples, when carbodiimide and hydroxysuccinimide were subjected to surface treatment with only one of them, sufficient reaction sensitivity was not obtained, and it was preferable to use both. In particular, as carbodiimide acts while hydroxysuccinimide is in contact with the carrier surface, for example, treating the hydroxysuccinimide solution in the order of adding the carbodiimide after supplying the hydroxysuccinimide solution to the carrier tends to further improve sensitivity. there were.

[Example 2] In this example, a platelet cross compatibility test using the antibody-sensitized magnetic particles prepared in Example 1 will be described.

First, about 150 mL of A corresponding to 15 vol% is added to about 1 mL of the platelet suspension obtained from the platelet preparation segment.
After adding and stirring the CD-A solution, centrifuge it and centrifuge.
(Plasma) was removed, and the cells were suspended in 10 mM EDTA-PBS (pH 6.0) so that the number of platelets became 30,000 / μL.

The prepared platelet suspension was dispensed at 50 μL / well into a WGA solid phase plate (manufactured by Olympus Optical Industry Co., Ltd.) on which WGA (wheat germ lectin) described in JP-A-2-124464 was immobilized. After centrifugation, the unsolidified platelets were washed and separated.

The patient's serum (containing each anti-HPA-1a antibody, anti-HPA-3a antibody, anti-HPA-4a antibody, and anti-HPA-5a antibody) was mixed with a low ionic strength buffer (LISS solution), and the platelets were fixed. 100 μL / well was dispensed into the plates that had been combined.

After incubation at 37 ° C. for 30 minutes, unreacted antibodies were separated by washing.

The suspension of about 0.5% magnetic gelatin particles prepared in Example 1 was dispensed at 25 μL / well.

The plate was left on a magnet for 2 minutes, and the generated distribution pattern in the well was confirmed.

Table 1 shows the results.

[0069]

[Table 1]

In the table, (-) indicates a negative pattern (unreacted), and a positive pattern indicates a dilution ratio of a positive patient serum.
(× 400, × 800).

As is clear from Table 1, platelets lacking the HPA-3a antigen did not bind to the anti-HPA-3a bioactive substance. On the other hand, platelets having HPA-1a antigen and HPA-4a antigen have anti-HPA-1a antibody and anti-H
PA-4a bioactive substance bound.

As described above, the magnetic gelatin particles prepared in Example 1 did not show non-specific adsorption.
It became clear that good test results were obtained.

Example 3 In this example, a platelet antibody screening test using the antibody-sensitized magnetic particles prepared in Example 1 will be described.

The positive control serum and the negative control serum in the kit of Anti-PLT / Olivio / MPHAII ^™ (manufactured by Olympus Optical Co., Ltd.) were diluted.

Subsequently, the platelet antigen plate was immersed in a washing container containing the washing solution in the kit for 5 minutes so as to completely immerse the inside of the well, and then washed five times. After soaking for 5 minutes, the plate was washed 5 times. The washed antigen plate was hit on a paper towel or the like to thoroughly remove the washing solution.

The diluted serum was dispensed at 25 μL / well,
The antigen plate was allowed to stand in a wet state at room temperature for 2 hours.

After the reaction, the specimen is discarded, the antigen plate is washed four times with the above-mentioned washing solution, the washing solution is exchanged, the antigen plate is washed once, and the washed antigen plate is hit on a paper towel or the like. Then, the washing solution was cut off.

The suspension of about 0.5% magnetic gelatin particles prepared in Example 1 was dispensed at 25 μL / well.

The antigen plate was allowed to stand on a magnet for 3 minutes, and the resulting distribution pattern in the well was confirmed. Table 2 shows the results.

[0080]

[Table 2]

In Table 2, the negative pattern (unreacted)
"-", Positive patterns are "+" (slightly weak), "++"
(Strong), "+++" (very strong).

As is clear from Table 2, when the negative control serum was used, no reaction was observed even at a dilution of 16 times, whereas when the positive control serum was used, the dilution was not.
The reaction was shown up to 256 times.

From the above, it was revealed that the use of the antibody-sensitized magnetic particles prepared in Example 1 enables a platelet antibody screening test to be performed with high accuracy.

[0084]

According to the solid phase of the present invention, a highly accurate immunological test can be achieved in a short time. Further, the solid phase of the present invention can be stably stored in a liquid.

[Brief description of the drawings]

FIG. 1 shows a cross-compatibility test of platelets using the solid phase of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) G01N 33/543 525 G01N 33/543 525U 525W 541 541A C07K 1/08 C07K 1/08 17/02 17/02 G01N 33/553 G01N 33/553

Claims (3)

[Claims] 1. A solid phase in which a biologically active substance is directly bound to a surface, wherein at least a part of the surface of the solid phase is chemically activated and another part is bound to the biologically active substance. A solid phase characterized by being non-interfering with the solid phase. 2. The solid phase according to claim 1, wherein the solid phase is a carrier having a suspending property and a sedimenting property in a liquid, and appropriately containing a magnetic substance. 3. Arranging a chemically activatable portion on at least a part of the surface and chemically activating a solid phase having a property of being non-interfering with the bioactive substance. A method for producing a solid phase having a biological activity, comprising: a step of bringing a bioactive substance into contact with the surface of the activated solid phase.