JP2002005936A - Measurement reagent and measurement based on enzyme immunoassay - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve problems, such as corrosion of an analysis apparatus and treatment of experiment waste liquid, and to provide the measurement reagent and the measurement for the substance to be measured in the sample based on the enzyme immunoassay with enhanced accuracy to completely the enzyme reaction without causing discoloration, by using a protein denaturant as a stop agent for an enzyme reaction by an enzyme label substance, in the case of a reagent or a measurement for measuring a substance to be measured in a sample based on an enzyme immunoassay. SOLUTION: The stop agent made up of the protein denaturant for the enzyme reaction by the enzyme label substance is contained in the reagent, or caused to exist in the measurement, for measuring the substance to be measured in the sample based on the enzyme immunoassay.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a measuring reagent and a measuring method for measuring a substance to be measured in a sample by an enzyme immunoassay, wherein a protein denaturing agent is used as a terminator for the enzyme reaction of a labeling substance. It is characterized by the following. The present invention is particularly useful in the fields of chemistry, life science, clinical testing, and the like.

[0002]

2. Description of the Related Art A trace amount contained in a sample utilizing a reaction between a substance having a specific affinity such as an antigen and an antibody, a sugar and a lectin, a nucleotide chain and its complementary nucleotide chain, and a ligand and a receptor. Various reagents and methods for measuring a target substance are known.

[0003] Among them, immunological measurement methods utilizing an antigen-antibody reaction (immune reaction) between an antigen and an antibody are widely practiced. Among the immunological measurement methods, by binding a label capable of distinguishing a minute difference such as an enzyme, a radioisotope, or a fluorescent dye to an antigen or an antibody, by utilizing the specificity of the antigen-antibody reaction, An immunological measurement method using a label for immunologically measuring the presence of an antigen or an antibody in a sample is widely used because it can be easily performed in a relatively short time and has good reproducibility.

[0004] Of the immunoassays using this label, those using enzymes as the label are enzyme immunoassays (EIA; Enzyme Immunoassay).
That. In this EIA, one of an antigen and an antibody is labeled with an enzyme, the result of the antigen-antibody reaction is converted into an enzymatic reaction to develop color, and the antigen or antibody is qualitatively or quantitatively measured. Further, the EIA method using a solid phase was used for the ELISA method (Enzyme-Linked Im).
(Munosorbent Assay). These enzyme immunoassays are widely used because they have the same sensitivity as radioimmunoassay using a radioisotope as a labeling substance and there is no danger of radioactive contamination.

[0005] In this enzyme immunoassay, a method for measuring the enzymatic activity of an enzyme-labeled substance using an optical method is generally well known. In this optical method, a dye or the like is generated from a chromogen by the catalytic activity of an enzyme as a labeling substance, and the enzyme activity can be determined by measuring the amount of the generated dye or the like. In the enzyme immunoassay, it is common to measure the absorbance after stopping the enzymatic reaction by the enzyme-labeled substance.

In order to stop the enzymatic reaction, a substance such as a strong acid such as sulfuric acid or hydrochloric acid, or a strong alkaline liquid such as sodium hydroxide is generally used.

However, these generally used terminating agents for enzyme reactions may corrode analytical instruments and also have problems such as treatment of experimental waste liquid. Needed to be careful. Furthermore, when peroxidase is used as the labeling substance, 4-aminoantipyrine and phenol or one of its derivatives or aniline derivatives are used as the chromogen, a strong acid such as sulfuric acid is used as a terminator for the enzyme reaction. When the method is used, discoloration occurs after the enzymatic reaction is stopped, and measurement becomes impossible.

The present invention has been made in view of the above problems, and solves problems such as corrosion of analytical instruments and treatment of experimental waste liquid by using a protein denaturant as a terminator of an enzyme reaction by an enzyme-labeled substance. Further, it is another object of the present invention to provide a reagent and a method for measuring a substance to be measured in a sample by an enzyme immunoassay method with improved accuracy by completely stopping the enzyme reaction without causing fading.

[0008]

SUMMARY OF THE INVENTION The present invention provides a reagent for measuring a substance to be measured in a sample by an enzyme immunoassay, which comprises a terminator for stopping an enzyme reaction by an enzyme labeling substance comprising a protein denaturing agent. A reagent for measuring a substance to be measured in a sample by an enzyme immunoassay, characterized in that:

In the measuring reagent of the present invention, the protein denaturant is preferably at least one of guanidine hydrochloride, urea and thiocyanate.

[0010] In the measurement reagent of the present invention, it is preferable that the labeling substance is peroxidase.

In the measuring reagent of the present invention, (i) 4-aminoantipyrine and (i)
i) It is preferable to use either phenol or a derivative thereof or an aniline derivative.

Further, in the measurement reagent of the present invention, it is preferable that the substance to be measured is an antibody against a microorganism causing summer-type hypersensitivity pneumonia.

In the measuring reagent of the present invention, the microorganism causing summer-type hypersensitivity pneumonia is Trichosporon.
Asahii is preferred.

The present invention also relates to a measuring method for measuring a substance to be measured in a sample by an enzyme immunoassay,
A method for measuring a substance to be measured in a sample by an enzyme immunoassay, which comprises using a protein denaturant as a terminator for an enzyme reaction by an enzyme-labeled substance.

In the measuring method of the present invention, the protein denaturant is preferably at least one of guanidine hydrochloride, urea and thiocyanate.

[0016] In the measurement method of the present invention, it is preferable that the labeling substance is peroxidase.

Further, in the measurement method of the present invention, (i) 4-aminoantipyrine and (i)
i) It is preferable to use either phenol or a derivative thereof or an aniline derivative.

In the measurement method of the present invention, it is preferable that the substance to be measured is an antibody against a microorganism causing summer-type hypersensitivity pneumonia.

Further, in the measurement method of the present invention, the microorganism causing summer-type hypersensitivity pneumonia is Trichosporon.
Asahii is preferred.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION A measuring reagent and a measuring method for a substance to be measured in a sample according to the present invention are a reagent for measuring a substance to be measured by an enzyme immunoassay and a measuring method. Accurate measurement can be performed by using a protein denaturant as a terminator.

In the measuring reagent and the measuring method of the present invention, the terminator of the enzyme reaction by the enzyme-labeled substance is changed from the conventionally used sulfuric acid or hydrochloric acid to a protein denaturing agent such as guanidine hydrochloride or urea, thereby reducing Can be accurately measured. In particular, it is possible to prevent fading after stopping the enzyme reaction and to perform accurate measurement.

In the measuring reagent and the measuring method of the present invention,
The procedure for measuring the substance to be measured in the sample may be performed according to the procedure of a known enzyme immunoassay.

The procedure of this operation will be described below, taking as an example the case where the substance to be measured is an antibody and measurement is performed by the sandwich method.

“A substance capable of binding to an antibody to be measured” (for example, an antigen for an antibody to be measured or a substance having an antigenic determinant of this antigen) is immobilized on a carrier. Prepare the let.

Next, a fixed amount of a sample is added to a portion of the carrier on which the “substance capable of binding to the antibody to be measured” is immobilized, and the carrier is contacted for a predetermined time.

In this way, when the substance to be measured is present in the sample, a bond of "substance to be measured-substance capable of binding to the antibody to be measured-carrier" is formed.

Simultaneously with the addition / contact, within a certain period of time, or after washing, the “substance capable of binding to the antibody to be measured” and the “substance capable of binding to the antibody to be measured” A fixed amount of an enzyme-labeled substance (“enzyme-labeled binding substance”), which is the same or different from a substance capable of binding (eg, an antibody or an antigen), is transferred to the “substance to be measured” of this carrier. -Substance capable of binding to the antibody to be measured "is added to the immobilized portion and contacted for a certain period of time.

By this operation, when the sample contains an antibody which is a substance to be measured, an enzyme-labeled binding substance-a substance to be measured-a substance capable of binding to the antibody to be measured-a carrier " Form a bond.

Thereafter, unbound “enzyme-labeled binding substance” not bound to the carrier is separated by washing. (B / F separation)

Next, a chromogen is added to the portion of “enzyme-labeled binding substance—substance to be measured—substance capable of binding to the antibody to be measured—carrier” of the carrier, and enzyme labeling (binding) is performed. A dye is produced from this chromogen by an enzymatic reaction with the substance.

Thereafter, a fixed amount of an enzyme reaction terminator based on an enzyme-labeled (binding) substance composed of a protein denaturant is added to the carrier to stop the enzyme reaction.

Then, by measuring the amount (concentration) of the produced dye, the carrier is bound by the binding of “enzyme-labeled binding substance—substance to be measured—substance capable of binding to the antibody to be measured—carrier”. The enzyme activity of the enzyme-labeled binding substance bound to is measured, whereby the measurement target substance in the sample is measured.

In this measurement, the carrier and the enzyme-labeled binding substance are referred to as “enzyme-labeled binding substance—measurement substance—substance capable of binding to the antibody to be measured—through the substance to be measured in the sample. By binding to the "carrier", the presence or absence or the amount of the substance to be measured contained in the sample can be measured by measuring the amount of the enzyme-labeled binding substance indirectly bound to the carrier.

For the measurement of the enzyme activity of the enzyme-labeled binding substance bound to the carrier by the binding of “enzyme-labeled binding substance-substance to be measured-substance capable of binding to the antibody to be measured-carrier”, for example, When peroxidase is used as the labeling substance and (i) 4-aminoantipyrine and (ii) any one of phenol or its derivative or aniline derivative are used as the chromogen, the reaction is carried out in the presence of hydrogen peroxide. Then, a leuco dye may be generated from the chromogen, and the amount of the generated leuco dye may be measured by an optical method such as measuring absorbance.

The terminator of the enzyme reaction used in the measuring method and the measuring reagent of the present invention comprises a protein denaturing agent. In the measurement method and the measurement reagent of the present invention, only a protein denaturant may be used as a terminator for an enzyme reaction, or a combination of a protein denaturant and an auxiliary component may be used as a terminator for an enzyme reaction. May be.

As the protein denaturing agent used as a reaction terminator in the measuring method and the measuring reagent of the present invention, for example, guanidine hydrochloride, urea or thiocyanate is preferably used. Here, when guanidine hydrochloride is used as the reaction terminator, the final concentration is 2 M or more,
Preferably, the enzyme reaction can be reliably stopped by adding so that the concentration becomes 4 M or more. When urea is used as a reaction terminator, the enzyme reaction can be surely stopped by adding urea to a final concentration of 2 M or more, preferably 4 M or more. Further, when thiocyanate is used as a reaction terminator, the enzyme reaction can be surely stopped by adding the thiocyanate to a final concentration of 2 M or more, preferably 4 M or more. The protein denaturant may be used alone or in combination of two or more.

The labeling substance used in the measuring method and the measuring reagent of the present invention includes, for example, peroxidase, alkaline phosphatase, β-galactosidase,
Luciferase and the like can be mentioned.

In the measuring method and the measuring reagent of the present invention, it is preferable to use peroxidase as a labeling substance. Here, as the peroxidase, for example, those derived from microorganisms such as bacteria or fungi, those derived from animals such as humans or cattle, those derived from plants such as horseradish, those prepared by genetic recombination, and the like are used. Can be. The concentration of peroxidase used was 160-640 purpurogallin mU / ml.
Is preferably used in the concentration range.

Examples of the chromogen used in the measuring method and the measuring reagent of the present invention include 4-aminoantipyrine and a combination of phenol or a derivative thereof or an aniline derivative, 3-methyl-2-benzothiazolinone hydrazone and aniline Combination of derivatives, 2,
2'-azinobis (3-ethylbenzothiazoline-6-
Sulfonic acid), triphenylmethane leuco dye, diphenylamine derivative, benzidine derivative, triallylimidazole derivative, leucomethylene blue derivative or ο
-Phenylenediamine derivatives and the like.

Here, as the phenol derivative, for example, 4-chlorophenol, 2,4-dichlorophenol, 2,4-dibromophenol, or 2,4,6
-Trichlorophenol or salts thereof.

As the aniline derivative, for example,
N- (2-hydroxy-3-sulfopropyl) -3,5
-Dimethoxyaniline (HDAOS), N-sulfopropyl-3,5-dimethoxyaniline (HDAPS),
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-
Ethyl-N-sulfopropyl-3,5-dimethoxyaniline (DAPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxy-4-fluoroaniline (FDAOS), N- Ethyl-N-sulfopropyl-3,5-dimethoxy-4-fluoroaniline (FDAPS), N- (2-carboxyethyl) -N
-Ethyl-3,5-dimethoxyaniline (CEDB),
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline (ADOS), N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline (ADPS), N-ethyl- N- (2-hydroxy-3
-Sulfopropyl) aniline (ALOS), N-ethyl-N- (3-sulfopropyl) aniline (ALPS),
N- (3-sulfopropyl) aniline (HALPS),
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (3-sulfopropyl) -3,5-dimethylaniline (MAPS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline (TOOS), N- (2-carboxyethyl) -N-ethyl-3-methylaniline (CEMB), or N-
Examples thereof include (2-carboxyethyl) -N-ethyl-3-methoxyaniline (CEMO), and salts thereof.

Examples of the triphenylmethane-based leuco dye include leucomalachite green and bis (p
-Diethylaminophenyl) -2-sulfonylmethane or bis (p-diethylaminophenyl) -3,4-disulfopropoxyphenylmethane disodium salt.

Further, as the diphenylamine derivative,
For example, bis [4-di (2-butoxyethyl) amino-
2-methylphenyl] amine or N, N-bis (4-)
Diethylamino-2-methylphenyl) -N'-p-toluenesulfonylurea and the like.

As the leucomethylene blue derivative, for example, sodium salt of 10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine or 10- [3- (methoxycarbonylaminomethyl) phenylmethylamino Carbonyl)-
3,7-bis (dimethylamino) phenothiazine and the like can be mentioned.

Further, benzidine derivatives include, for example, benzidine, o-tolidine, o-dianisidine,
3,3′-diaminobenzidine or 3,3 ′, 5,5 ′
-Tetraaminobenzidine and the like.

As the triallylimidazole derivative, for example, 2- (4-carboxyphenyl) -3-
N-methylcarbamoyl-4,5-bis (4-diethylaminophenyl) imidazole or 2- (3-methoxy-4-diethylaminophenyl) -3-N-methylcarbamoyl-4,5-bis (2-methyl-4- (Diethylaminophenyl) imidazole and the like.

In the measuring method and the measuring reagent of the present invention, it is preferable to use (i) 4-aminoantipyrine and (ii) phenol or a derivative thereof or an aniline derivative as a chromogen. .

Regarding the concentration of the chromogen used, 4-
Aminoantipyrine is preferably used in a concentration range of 0.1 to 10 mM, and phenol or a derivative thereof or an aniline derivative is preferably used in a concentration range of 6 to 300 mM.

The measuring method and the measuring reagent of the present invention can be performed by, for example, a sandwich method or a competitive method.

Further, the measuring method and the measuring reagent of the present invention include:
For example, it can be performed by a method such as a one-step method or a two-step method.

In the measuring method and the measuring reagent of the present invention, a substance made of a magnetic substance or a substance containing a magnetic substance is used as a carrier, and a magnetic force is applied to the substance to magnetically perform B / F separation. Can also.

In the measuring method and the measuring reagent of the present invention,
As the carrier, any carrier, on which an antigen or an antibody can be immobilized, can be used without any particular limitation on its type, material, shape and the like.

This is because the carrier generally used for the measurement in the immunoassay using a labeling substance such as enzyme immunoassay, fluorescence immunoassay, radioimmunoassay, or luminescence immunoassay is used. Can be used without any problem.

For example, polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate,
Polyacrylamide, latex, liposome, gelatin, agarose, cellulose, sepharose, glass,
Particles made of materials such as metals, ceramics or magnetic materials,
Carriers such as microcapsules, beads, microplates, test tubes, sticks or test pieces can be used.

It is also possible to use a magnetic carrier prepared by coating the above-mentioned carrier with a ferromagnetic material or incorporating a ferromagnetic material when molding the carrier.

A known method such as a chemical bonding method can be used to bind the enzyme-labeled substance to an antigen or an antibody.

In the case of performing by this chemical bonding method, the Japanese Society of Clinical Pathology, “Special Issue on Clinical Pathology Extraordinary Issue No. 53, Immunoassay for Clinical Examination-Technology and Application-”, Clinical Pathology Publishing Association, 1983, Japan The Biochemical Society of Japan, “Course for New Chemistry Experiment 1 Protein IV”, Tokyo Kagaku Dojin, 1991
According to a known method described in the year and the like, an enzyme-labeled substance, an antigen or an antibody, glutaraldehyde, carbodiimide,
Mixing and contacting with a divalent crosslinking reagent such as imide ester or maleimide, and binding by reacting the enzyme labeling substance with the respective amino group, carboxyl group, SH group, aldehyde group or hydroxyl group of antigen or antibody etc. It can be performed.

As a method for immobilizing an antigen or an antibody on a carrier, a known method such as a physical adsorption method or a chemical bonding method can be used.

In the case of the physical adsorption method, according to a known method, an antigen or antibody dissolved in a buffer or the like is added to and contacted with a portion of the carrier to be immobilized, or the antigen or antibody is contacted with the carrier. , A buffer solution or the like, and mixing.

For example, an antigen or antibody dissolved in a buffer solution or the like is added to a portion of the carrier to be immobilized and brought into contact,
Alternatively, the antigen or antibody and the carrier are mixed and brought into contact with each other in a solution such as a buffer solution, and this is carried out at about 2 ° C. to about 40 ° C. for about 10 minutes to about 1 day.

In the case where the chemical bonding method is used,
The Japanese Society of Clinical Pathology “Clinical Pathology Special Issue No. 53”
Immunoassay for Clinical Examination -Technology and Application- ", Clinical Pathology Publishing Association, 1983, edited by The Biochemical Society of Japan," New Biochemistry Experiment Course 1 Protein IV ", Tokyo Kagaku Dojin, 199
According to a known method described in one year or the like, a carrier or a portion to be immobilized on a carrier, an antigen or an antibody, and the like are mixed and contacted with a divalent crosslinking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide, The immobilization can be carried out by reacting with a carrier, a portion to be immobilized on the carrier, or an amino group, a carboxyl group, an SH group, an aldehyde group, a hydroxyl group, or the like of an antigen or an antibody.

Further, if it is necessary to carry out a treatment in order to suppress a non-specific reaction, the immobilized portion of the carrier on which the antigen or the antibody is immobilized may be replaced with BSA, casein, gelatin, ovalbumin or its ovalbumin. Proteins such as salt,
The blocking treatment (masking treatment) may be performed by a known method such as contacting and coating a surfactant or skim milk powder or the like.

As a solution for dissolving an antigen or antibody bound with an enzyme-labeled substance, a substance to be measured, or the like, or a diluent of a sample, various aqueous solvents can be used.

For example, aqueous solvents such as purified water, physiological saline or various buffers such as tris (hydroxymethyl) aminomethane buffer, phosphate buffer or phosphate buffered saline can be used. The buffer p
H is preferably in the range of pH 3 to 12.

As the buffer, for example, MES, B
is-Tris, Bis-Tris propane, ADA,
PIPES, ACES, MOPSO, MOPS, BE
S, TES, HEPES, DIPSO, TAPSO, P
OPSO, HEPES, HEPPSO, EPPS, Tr
aqueous solution of a buffer such as icine, Bicine, TAPS, CHES, phosphoric acid, phosphate, boric acid, borate, glycine, glycylglycine, imidazole, or tris (hydroxymethyl) aminomethane [Tris]. be able to.

In addition, BSA, human serum albumin, BSA, human serum albumin, etc.
Proteins such as casein or its salts, various salts such as sodium chloride, various saccharides, skim milk powder, various animal sera such as normal rabbit serum, body fluids of immune animals or components thereof, components derived from genetically modified host organisms, azide Various preservatives such as sodium or various surfactants such as a nonionic surfactant, an amphoteric surfactant or an anionic surfactant can be appropriately added and used.

The concentration when these are added is not particularly limited, but may be 0.001 to 10% (W /
V) is preferable, and particularly preferably 0.01 to 5% (W / V).

The surfactants 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, and polyoxyethylene alkyl ether. , Polyoxyethylene phytosterol, phytostanol, polyoxyethylene polyoxypropylene alkyl ether,
Non-ionic surfactants such as polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil or polyoxyethylene lanolin, amphoteric surfactants such as betaine acetate, or polyoxyethylene alkyl ether sulfate or polyoxyethylene Anionic surfactants such as alkyl ether acetates can be mentioned.

The substance to be measured in the measuring reagent and the measuring method of the present invention may be any biologically relevant substance such as an organic substance such as protein, carbohydrate, lipid and nucleic acid, and an inorganic substance. Specifically, bacteria-related antigens or antibodies such as antibodies against the causative microorganism of summer-type hypersensitivity pneumonia, Escherichia coli O157 antigen, anti-Treponemal pallidum (TP) antibody, anti-mycoplasma antibody, and anti-streptolidine O antibody (ASO); HBs antigen, anti-HBs antibody, HBe antigen, anti-HBe antibody, anti-HBc antibody, anti-HCV antibody, anti-H
Virus-related antigens or antibodies such as an IV antibody and an anti-ATLV antibody; immunoglobulin G (IgG), immunoglobulin A
Immunoglobulin such as (IgA), immunoglobulin M (IgM) or immunoglobulin E (IgE); C-reactive protein (CRP), α1-acid glycoprotein, haptoglobin, complement C3, complement C4, rheumatoid factor, etc. Inflammatory markers; α-fetoprotein, CEA, C
Tumor markers such as A19-9; hormones such as human placental chorionic gonadotropin; allergens, allergen-specific Igs
Allergen-related antigens or antibodies such as E antibody; blood coagulation-related substances such as antithrombin III (ATIII); fibrinolytic substances (FDP); fibrinolysis-related substances such as D-dimer; ABO blood group antibodies, irregular Blood group-related antigens or antibodies such as antibodies; viral DNA or RNA; bacterial DNA or RNA; human or other animal or plant DNs
A or RNA; substances associated with other diseases such as lipoprotein (a), ferritin; drugs; metals; toxic substances or deleterious substances.

In the measuring reagent and the measuring method of the present invention, the substance to be measured is preferably an antibody against a microorganism causing summer-type hypersensitivity pneumonia. In particular, in the measurement reagent and the measurement method of the present invention, the substance to be measured is "Trichosporo", which is a microorganism causing summer-type hypersensitivity pneumonia.
Preferably, the antibody is against "nasahi".

In the present invention, a sample refers to a liquid that has a possibility that the above-mentioned substance to be measured is present and that is to be used for confirming the presence or absence of the substance to be measured or, in some cases, performing quantification.

For example, human or animal blood, serum, plasma, urine, semen, cerebrospinal fluid, saliva, sweat, tears, ascites, amniotic fluid, etc .; human or animal brain and other organs, hair, skin, nails, etc.
Extract of muscle or nervous tissue, etc .; Extract or suspension of human or animal feces; Extract of cells or bacteria; Extract of plants; Cereals, vegetables, fruits, seafood, meat, processed foods, etc. Food, water, tea, coffee, milk, or beverages such as fruit juice,
Examples include environmental analysis samples such as drinking water, river water, lake water, seawater, and soil suspension.

[0073]

[Comparative example]

Using sulfuric acid which has been conventionally used as a terminator for the enzyme reaction, the effect of terminating the enzyme reaction when IgG in human serum samples was measured was confirmed.

(1) Preparation of Reagent Preparation of Phosphate Isotonic Buffer Buffer In about 80 ml of purified water, 0.29 g of disodium hydrogen phosphate / decahydrate, 0.02 g of potassium dihydrogen phosphate, sodium chloride 0.8 g of potassium chloride and 0.02 of potassium chloride
g was added and dissolved, and the total volume was adjusted to 100 ml with purified water to prepare a phosphate isotonic buffer.

Preparation of Blocking Solution About 80 m of purified water
In 0.1, 0.1 of tris (hydroxymethyl) aminomethane is added.
After adding and dissolving 605 g and 0.88 g of sodium chloride,
The pH was adjusted to 8.3 by adding hydrochloric acid. Furthermore, after adding and dissolving 0.5 g of sodium caseinate and 0.2 g of sodium azide, the total volume was adjusted to 100 ml with purified water.

Preparation of Washing Solution In about 80 ml of purified water, 0.29 g of disodium hydrogen phosphate / decahydrate, 0.02 g of potassium dihydrogen phosphate,
0.8 g of sodium chloride, 0.02 of potassium chloride
g, Tween 20 (surfactant) was added and dissolved, and the total volume was adjusted to 100 ml with purified water.

Dilution of POD-labeled antibody In about 80 ml of purified water, 0.605 g of tris (hydroxymethyl) aminomethane and 0.88 g of sodium chloride were added.
g, lactose / monohydrate (1.0 g) was added and dissolved,
The pH was adjusted to 8.0 by adding hydrochloric acid. Furthermore, 0.5 g of sodium caseinate and 0.01 g of gentamicin sulfate
Was added and dissolved, and the total volume was adjusted to 100 ml with purified water.

(2) Measurement for Examining the Effect of Terminating Enzyme Reaction Human serum having an IgG concentration of 1,000 mg / dl was diluted with a phosphate isotonic buffer, and the IgG concentration was 100 ng each.
/ Ml and 1000 ng / ml. Purified water was used as a reagent blank sample.
These were dispensed into the wells of the microplate at 100 μl each, and then allowed to stand overnight at 4 ° C. to immobilize human IgG on the inner wall of each well.

Next, the solution in the wells of the microplate was removed, and the microplate was washed twice with a washing solution. Thereafter, 200 μl of the blocking solution was dispensed into each well to perform blocking on each well. Then, the solution in the well of the microplate was removed, and the well was washed twice with a washing solution. The one prepared by the above operation was used as a human IgG-binding microplate.

(2) Measurement for Examining the Effect of Terminating Enzyme Reaction Each well of the human IgG-binding microplate was
POD diluted 2,000-fold with POD-labeled antibody diluent
Labeled anti-human IgG antibody solution (manufactured by Sigma; A017)
0, Fc specific) in each well 100 μl
The reaction was continued at 37 ° C. for 1 hour. Next, each well was washed with a washing solution.

Thereafter, the chromogen solution [1 mM 4-aminoantipyrine, 20 mM phenol, and 0.006%
0.1 M Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer (pH 8.0) containing hydrogen peroxide] was added to each well, and the mixture was reacted at 37 ° C. for 1 hour.

After this reaction, 4
An aqueous solution of N sulfuric acid was added to each well. In addition, a control obtained by adding purified water instead of the 4N aqueous sulfuric acid solution was used as a control. The amounts of the chromogen solution and the 4N sulfuric acid aqueous solution or purified water used as a terminator for the enzymatic reaction were as follows. a) Sulfuric acid final concentration 0.8 M: 100 μl of 4N sulfuric acid aqueous solution with respect to 150 μl of chromogen solution b) Control: 150 μl of chromogen solution with purified water 100
μl Then, immediately after the addition of a 4N aqueous sulfuric acid solution or purified water using a microplate reader (manufactured by Bio-Rad; type 3550), 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, and After 60 minutes, the absorbance (main wavelength 490 nm, auxiliary wavelength 655 nm) of each well was measured.

(3) Result of stopping enzyme reaction Absorbance of purified water sample (reagent blank) and human IgG
Table 1 shows the absorbances of the samples (two types) after subtracting the absorbance of the reagent blank.

[0085]

[Table 1]

In Table 1 showing the measurement results of the human IgG sample (two types) and the purified water sample (reagent blank), the discoloration of the sample to which the sulfuric acid aqueous solution was added was immediately observed as compared with the sample to which purified water was added. It turns out that measurement has become impossible. In the case of adding purified water instead of the sulfuric acid aqueous solution (control), no fading occurred, but the absorbance increased with time, indicating that the enzyme reaction had progressed. .

[0087]

EXAMPLES The present invention will now be described specifically with reference to examples, but the present invention is not limited thereto.

[Example 1]

Using guanidine hydrochloride as a terminator for the enzyme reaction according to the present invention, the effect of terminating the enzyme reaction when IgG in human serum samples was measured was confirmed.

(1) Preparation of Reagent Preparation of Phosphate Isotonic Buffer Solution In about 80 ml of purified water, 0.29 g of disodium hydrogen phosphate / decahydrate, 0.02 g of potassium dihydrogen phosphate, sodium chloride 0.8 g of potassium chloride and 0.02 of potassium chloride
g was added and dissolved, and the total volume was adjusted to 100 ml with purified water to prepare a phosphate isotonic buffer.

Preparation of Blocking Solution In about 80 ml of purified water, 0.605 g of tris (hydroxymethyl) aminomethane and 0.88 g of sodium chloride
Was added and dissolved, and hydrochloric acid was added to adjust the pH to 8.3. Further, after adding and dissolving 0.5 g of sodium caseinate and 0.2 g of sodium azide, a total amount of 1
00 ml.

Preparation of Washing Solution In about 80 ml of purified water, 0.29 g of disodium hydrogen phosphate / decahydrate, 0.02 g of potassium dihydrogen phosphate,
0.8 g of sodium chloride, 0.02 of potassium chloride
g, Tween 20 (surfactant) was added and dissolved, and the total volume was adjusted to 100 ml with purified water.

POD-Labeled Antibody Dilution In about 80 ml of purified water, 0.605 g of tris (hydroxymethyl) aminomethane and 0.88 g of sodium chloride were added.
g, lactose / monohydrate (1.0 g) was added and dissolved,
The pH was adjusted to 8.0 by adding hydrochloric acid. Furthermore, 0.5 g of sodium caseinate and 0.01 g of gentamicin sulfate
Was added and dissolved, and the total volume was adjusted to 100 ml with purified water.

(2) Measurement for Examining the Effect of Terminating Enzyme Reaction Human serum having an IgG concentration of 1,000 mg / dl was diluted with a phosphate isotonic buffer, and each IgG concentration was 100 ng.
/ Ml and 1000 ng / ml. Purified water was used as a reagent blank sample.
These were dispensed into the wells of the microplate at 100 μl each, and then allowed to stand overnight at 4 ° C. to immobilize human IgG on the inner wall of each well.

Next, the solution in the wells of the microplate was removed, and the microplate was washed twice with a washing solution. Thereafter, 200 μl of the blocking solution was dispensed into each well to perform blocking on each well. Then, the solution in the well of the microplate was removed, and the well was washed twice with a washing solution. The one prepared by the above operation was used as a human IgG-binding microplate.

To each well of the human IgG-binding microplate, 100 μl of a POD-labeled anti-human IgG antibody solution (manufactured by Sigma; A0170, Fc specific) diluted 5,000-fold with a POD-labeled antibody diluent was added.
The reaction was carried out at 37 ° C. for 1 hour. Next, each well was washed with a washing solution.

Thereafter, the chromogen solution [1 mM 4-aminoantipyrine, 20 mM phenol, and 0.006%
0.1 M Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer (pH 8.0) containing hydrogen peroxide] was added to each well, and the mixture was reacted at 37 ° C. for 1 hour.

After this reaction, an aqueous solution of guanidine hydrochloride, which is a terminator for the enzyme reaction of the present invention, is added to a final concentration of 2M,
M and 5.3 M were added to each well. Also,
A control to which purified water was added instead of the guanidine hydrochloride aqueous solution was used as a control. The amount of each of the chromogen solution and the aqueous solution of guanidine hydrochloride or purified water used as a terminator for the enzyme reaction was determined as follows according to the final concentration of guanidine hydrochloride. a) Guanidine hydrochloride final concentration 2 M: Chromogen solution 150 μl
B) 150 μl of 4 M guanidine hydrochloride aqueous solution b) Guanidine hydrochloride final concentration 4 M: chromogen solution 150 μl
150 μl of 8 M guanidine hydrochloride aqueous solution c) Guanidine hydrochloride final concentration 5.3 M: chromogen 100
200 μl of 8 M guanidine hydrochloride aqueous solution per μl.
μl Then, using a microplate reader (manufactured by Bio-Rad; type 3550), immediately after the addition of an aqueous solution of guanidine hydrochloride or purified water at each concentration, 10 minutes, 20 minutes, 30 minutes, 40 minutes, and 50 minutes After 60 minutes, the absorbance (main wavelength 490 nm, sub-wavelength 655 nm) of each well was measured.

(3) Termination effect of enzyme reaction Absorbance of purified water sample (reagent blank) and human IgG
With respect to the samples (two types), the measurement results of the absorbance after subtracting the absorbance of the reagent blank are shown in Tables 2 and 3.

[0100]

[Table 2]

[0101]

[Table 3]

In Tables 2 and 3 showing the measurement results of the human IgG sample (two types) and the purified water sample (reagent blank), the final concentration of guanidine hydrochloride when an aqueous solution of guanidine hydrochloride was added as a terminator for the enzyme reaction. Is 2M, 4
M and 5.3M, the absorbance did not change or changed only slightly even after 60 minutes from the addition of the terminator, indicating that the progress of the enzyme reaction could be stopped. . In particular, the final concentration of guanidine hydrochloride is 4M
In the above cases, the effect is remarkable. In other words, it can be seen that even if the absorbance is measured after the elapse of time after the addition of the terminator, the substance to be measured can be accurately measured. On the contrary,
In the case of adding purified water in place of guanidine hydrochloride (control), the absorbance increased with time, indicating that the enzyme reaction had progressed.

When an aqueous solution of guanidine hydrochloride is added as a terminator for the enzymatic reaction, the absorbance of a reagent blank in which the sample is water is small, and this absorbance is also stable for a long time, so that accurate measurement of the substance to be measured is possible. It turns out to be suitable.

Thus, in the measurement according to the present invention, the progress of the enzyme reaction can be completely stopped by using guanidine hydrochloride as a terminator for the enzyme reaction, and discoloration after stopping the enzyme reaction can be prevented. It can be seen that accurate measurement can be performed.

[Example 2]

Using urea as a terminator for the enzyme reaction according to the present invention, the effect of terminating the enzyme reaction when IgG in human serum samples was measured was confirmed.

(1) Preparation of reagent

Reagents were prepared with the same reagent components and concentrations as in Example 1.

(2) Measurement for studying the effect of terminating the enzyme reaction Human serum having an IgG concentration of 1,000 mg / dl was diluted with a phosphate isotonic buffer, and the IgG concentration was 100 ng each.
/ Ml and 1000 ng / ml. Purified water was used as a reagent blank sample.
These were dispensed into the wells of the microplate at 100 μl each, and then allowed to stand overnight at 4 ° C. to immobilize human IgG on the inner wall of each well.

Next, the solution in the wells of the microplate was removed, and the microplate was washed twice with a washing solution. Thereafter, 200 μl of the blocking solution was dispensed into each well to perform blocking on each well. Then, the solution in the well of the microplate was removed, and the well was washed twice with a washing solution. The one prepared by the above operation was used as a human IgG-binding microplate.

To each well of the above human IgG-binding microplate, 100 μl of a POD-labeled anti-human IgG antibody solution (manufactured by Sigma; A0170, Fc specific) diluted 5,000-fold with a POD-labeled antibody diluent was added.
The reaction was carried out at 37 ° C. for 1 hour. Next, each well was washed with a washing solution.

Thereafter, the chromogen solution [1 mM 4-aminoantipyrine, 20 mM phenol, and 0.006%
0.1 M Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer (pH 8.0) containing hydrogen peroxide] was added to each well, and the mixture was reacted at 37 ° C. for 1 hour.

After this reaction, an aqueous urea solution, which is a terminator for the enzyme reaction of the present invention, is added to a final concentration of 2M, 4M, 5.3.
M was added to each well. In addition, a control obtained by adding purified water instead of the urea aqueous solution was used as a control. In addition,
The amount of each of the chromogen solution and the aqueous urea solution or purified water used as a terminator for the enzyme reaction was determined as follows according to the final concentration of urea. a) Urea aqueous solution final concentration 2M: 4M urea aqueous solution 150µl per 150µl of chromogen solution b) Urea aqueous solution final concentration 4M: 8M urea aqueous solution 150µl per 150µl chromogenic solution c) Final urea aqueous solution concentration 5. 3M: Chromogen solution 100 μl
D) control: 150 μl of chromogen solution and 150 μl of purified water
μl Then, using a microplate reader (manufactured by Bio-Rad; type 3550), immediately after the addition of urea aqueous solution or purified water of each concentration, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, After 60 minutes, the absorbance (main wavelength 490 nm, sub-wavelength 655 nm) of each well was measured.

(3) Termination effect of enzyme reaction Absorbance of purified water sample (reagent blank) and human IgG
Tables 4 and 5 show the measurement results of the absorbance of the samples (two types) after subtracting the absorbance of the reagent blank.

[0115]

[Table 4]

[0116]

[Table 5]

In Tables 4 and 5 showing the measurement results of the human IgG samples (two types) and the purified water samples (reagent blanks), the results obtained by adding an aqueous urea solution as a terminator for the enzyme reaction were as follows. When the concentration is 2M, 4M,
At any of 5.3M, the absorbance did not change or changed only slightly after 60 minutes from the addition of the terminator, indicating that the progress of the enzyme reaction was able to be stopped. In particular, when the final concentration of the urea aqueous solution is 4 M or more, the effect is remarkable. In other words, it can be seen that even if the absorbance is measured after a lapse of time after the addition of the terminator, the substance to be measured can be accurately measured. On the other hand, in the case of adding purified water instead of the urea aqueous solution (control),
The absorbance increased with time, indicating that the enzyme reaction had progressed.

When an aqueous urea solution is added as a terminator for the enzyme reaction, the absorbance of the reagent blank in which the sample is water is small, and the absorbance is stable for a long time, which is suitable for accurate measurement of the substance to be measured. You can see that it is.

As a result, in the measurement according to the present invention, the progress of the enzymatic reaction can be completely stopped by using urea as a terminator for the enzymatic reaction, and discoloration after stopping the enzymatic reaction can be prevented. It can be seen that the measurement can be performed easily.

[Embodiment 3]

A mouse monoclonal antibody (Ig) using thiocyanate as a terminator for the enzyme reaction according to the present invention was used.
The effect of stopping the enzyme reaction when G) was measured was confirmed.

(1) Preparation of measurement reagent Preparation of phosphate isotonic buffer solution In about 80 ml of purified water, 0.29 g of disodium hydrogen phosphate / decahydrate, 0.02 g of potassium dihydrogen phosphate, chloride 0.8 g of sodium and 0.02 of potassium chloride
g was added and dissolved, and the total volume was adjusted to 100 ml with purified water to prepare a phosphate isotonic buffer.

Preparation of Blocking Solution A solution of 10 mM monopotassium phosphate / borax buffer and 0.88 g of sodium chloride was added and dissolved in about 80 ml of purified water, and the pH was adjusted to 8.3 by adding hydrochloric acid. Further, 0.5 g of sodium caseinate and 0.1 g of sodium azide.
After adding and dissolving 2 g, the total volume was adjusted to 100 ml with purified water.

Preparation of Sample Diluent In about 80 ml of purified water, 0.605 g of tris (hydroxymethyl) aminomethane and 11.68 of sodium chloride were added.
g was added and dissolved, and hydrochloric acid was added to adjust the pH to 8.0. Further, after adding and dissolving 0.5 g of sodium caseinate and 0.2 g of sodium azide, a total amount of 1
00 ml.

Preparation of Washing Solution In about 80 ml of purified water, 0.29 g of disodium hydrogen phosphate / decahydrate, 0.02 g of potassium dihydrogen phosphate, 0.8 g of sodium chloride, 0.02 g of potassium chloride,
Tween 20 (surfactant) was added and dissolved in 0.05 g, and the total volume was adjusted to 100 ml with purified water.

POD-Labeled Antibody Diluent In about 80 ml of purified water, 0.605 g of tris (hydroxymethyl) aminomethane and 0.88 g of sodium chloride were added.
g, lactose / monohydrate (1.0 g) was added and dissolved,
The pH was adjusted to 8.0 by adding hydrochloric acid. Furthermore, 0.5 g of sodium caseinate and 0.01 g of gentamicin sulfate
Was added and dissolved, and the total volume was adjusted to 100 ml with purified water.

Preparation of rabbit anti-mouse IgG antibody binding microplate

A purified rabbit anti-mouse IgG antibody (Capp
el, ICN, 55480F, 1.0 mg / ml)
1 μg / ml was added to the phosphate isotonic buffer. This was dispensed to each well of the microplate in an amount of 100 μl each, and then left at 4 ° C. overnight to immobilize a rabbit anti-mouse IgG antibody on the inner wall of each well.

Next, the solution in the wells of the microplate was removed, and the microplate was washed twice with a washing solution. Thereafter, 200 μl of the blocking solution was dispensed into each well to perform blocking on each well. Then, the solution in the well of the microplate was removed, and the well was washed twice with a washing solution. The one prepared by the above operation was used as a rabbit anti-mouse IgG antibody binding microplate.

(2) Preparation of Sample A sample diluent was added to a purified mouse IgG monoclonal antibody stock solution having an IgG concentration of 1.6 mg / ml, and IgG was added.
The concentrations were 100 ng / ml, 200 ng / ml, 40
It was adjusted to be 0 ng / ml or 3200 ng / ml. Purified water was used as a reagent blank sample.

(3) Measurement 100 μl of each of the mouse IgG monoclonal antibody samples (four types) and the purified water sample (reagent blank) were added to each well of a rabbit anti-mouse IgG antibody binding microplate, and the mixture was added at 37 ° C. for 2 hours. Allowed to react for hours.

After washing these wells with a washing solution, a POD-labeled anti-mouse IgG antibody solution (manufactured by DAKO; diluted 1,000-fold with a POD-labeled antibody diluent) was used.
P-0161) was added to each well in an amount of 100 μl, and 3
The reaction was performed at 7 ° C. for 1 hour. Next, each well was washed with a washing solution.

Thereafter, the chromogen solution [1 mM 4-aminoantipyrine, 20 mM phenol, and 0.006%
0.1 M Tris (hydroxymethyl) aminomethane-hydrochloric acid buffer (pH 8.0) containing hydrogen peroxide] was added to each well, and the mixture was reacted at 37 ° C. for 1 hour.

After this reaction, an aqueous solution of sodium thiocyanate, which is a terminator for the enzyme reaction of the present invention, was added to each well to a final concentration of 2M, 4M, 5.3M. Further, a control obtained by adding purified water instead of the aqueous solution of sodium thiocyanate was used as a control. The amount of each of the chromogen solution and the aqueous solution of sodium thiocyanate or purified water used as a terminator for the enzyme reaction was determined as follows according to the final concentration of sodium thiocyanate. a) Sodium thiocyanate aqueous solution final concentration 2 M: 150 μl of chromogen solution 150 μl of 4 M sodium thiocyanate aqueous solution b) Final concentration of sodium thiocyanate aqueous solution 4 M: 150 μl of 8 M sodium thiocyanate aqueous solution with respect to 150 μl chromogen solution c) Sodium thiocyanate aqueous solution final concentration 5.3 M:
200 μl of an 8 M aqueous solution of sodium thiocyanate with respect to 100 μl of the chromogen solution, and then 10 minutes after the addition of an aqueous solution of sodium thiocyanate of each concentration or purified water using a microplate reader (Bio-Rad; type 3550). Absorbance of each well after 20 minutes, 30 minutes, 40 minutes, 50 minutes, and 60 minutes (primary wavelength 490 nm, secondary wavelength 655 n
m) was measured.

(3) Stopping effect of enzyme reaction Absorbance of purified water sample (reagent blank) and mouse Ig
With respect to the G monoclonal antibody samples (four types), the measurement results of the absorbance after subtracting the absorbance of the reagent blank are shown in Tables 6 and 7.

[0136]

[Table 6]

[0137]

[Table 7]

A mouse IgG monoclonal antibody sample (4
In Tables 6 and 7, which show the measurement results of the sample solution and the purified water sample (reagent blank), in the case where an aqueous solution of sodium thiocyanate was added as a terminator for the enzyme reaction, the final concentration of the aqueous solution of sodium thiocyanate was 2 M,
At 4M and 5.3M, 60 hours after the addition of the terminator.
Even after a lapse of minutes, the absorbance did not change or changed only slightly, indicating that the progress of the enzyme reaction could be stopped. In particular, when the final concentration of sodium thiocyanate is 4 M or more, the effect is remarkable. That is,
It can be seen that even when the absorbance is measured after a lapse of time after the addition of the terminator, the substance to be measured can be accurately measured. On the other hand, in the case where purified water was added instead of the aqueous solution of sodium thiocyanate (control), the absorbance increased with time, indicating that the enzyme reaction had progressed.

When an aqueous solution of sodium thiocyanate is added as a terminator for the enzymatic reaction, the value of the absorbance of the reagent blank in which the sample is water and its change are small, which is suitable for accurate measurement of the substance to be measured. You can see that there is.

Thus, in the measurement according to the present invention, the progress of the enzymatic reaction can be completely stopped by using an aqueous solution of sodium thiocyanate as a terminating agent for the enzymatic reaction, and discoloration after stopping the enzymatic reaction can be prevented. However, it can be seen that accurate measurement can be performed.

From the above, it was confirmed that the measuring reagent and the measuring method of the present invention can prevent the color fading after stopping the enzyme reaction and can perform the accurate measurement. Thus, it was confirmed that the measurement reagent and the measurement method can accurately diagnose a disease.

According to the measuring reagent and measuring method of the present invention, the terminator of the enzyme reaction by the enzyme-labeled substance is changed from the conventionally used sulfuric acid or hydrochloric acid to a protein denaturant such as guanidine hydrochloride, urea or thiocyanate. By changing, the substance to be measured in the sample can be accurately measured. In particular, it can completely stop the progress of the enzymatic reaction, prevent discoloration after stopping the enzymatic reaction, and perform accurate measurement. Further, in the measuring reagent and the measuring method of the present invention, the use of a protein denaturing agent as a terminator for an enzyme reaction by an enzyme-labeled substance can solve problems such as corrosion of analytical instruments and treatment of experimental waste liquid. .

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) C12R 1: 645) C12R 1: 645)

Claims (12)

[Claims] 1. A reagent for measuring a substance to be measured in a sample by an enzyme immunoassay, wherein the reagent comprises a terminator for an enzyme reaction by an enzyme labeling substance comprising a protein denaturing agent. A reagent for measuring a substance to be measured in a sample by a measurement method. 2. The method according to claim 1, wherein the protein denaturant is at least one of guanidine hydrochloride, urea and thiocyanate.
The measurement reagent according to the above. 3. The labeling substance is peroxidase,
The measurement reagent according to claim 1 or 2. 4. The chromogen according to claim 1, wherein (i) 4-aminoantipyrine and (ii) one of phenol or a derivative thereof and an aniline derivative are used. The measurement reagent according to any one of the preceding claims. 5. The measurement reagent according to claim 1, wherein the substance to be measured is an antibody against a microorganism that causes summer-type hypersensitivity pneumonia. 6. The microorganism causing summer-type hypersensitivity pneumonia is Tri.
The measuring reagent according to claim 5, which is chosporon asahii. 7. An enzyme-linked immunosorbent assay, wherein a protein denaturing agent is used as a terminator of an enzyme reaction by an enzyme-labeled substance in a measurement method for measuring a substance to be measured in a sample by an enzyme-linked immunosorbent assay. Method for measuring a substance to be measured in a sample by the method. 8. The method according to claim 7, wherein the protein denaturant is at least one of guanidine hydrochloride, urea and thiocyanate.
The measurement method described. 9. The labeling substance is peroxidase,
The measurement method according to claim 7. 10. The method according to claim 7, wherein (i) 4-aminoantipyrine and (ii) one of phenol or a derivative thereof and an aniline derivative are used as the chromogen. The measurement method according to any one of the preceding claims. 11. The method according to claim 7, wherein the substance to be measured is an antibody against a microorganism that causes summer-type hypersensitivity pneumonia. 12. The microorganism causing causative summer-type pneumonia is Tr.
12. Ichosporonasahii.
The measurement method described.