JPH1010127A - Aggregation immunoassay method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an aggregation immunoassay method, which can measure the antigen material in a sample under test in high measuring sensitivity by the aggregation of macromolucular carrier by using the monoclonal antibody, which is obtained by simple operation in the equal quantity. SOLUTION: The antigen material (e.g. glycohemoglobin A1c) in a sample under test is adsorbed or bonded to a macromolecular carrier (e.g. latex particles). Thereafter, at least one kind of the material, which is selected from the group comprising monoclonal antibody (e.g. antiglycohemoglobin A1c monoclonal antibody) that specifically reacts with the antigen material and protein A and protein-A like material and protein G and protein-G like material that are selectively bonded to this monoclonal antibody, is made to react furthermore. Thus, the macromolecular carrier is selective aggregated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for immunologically measuring an antigenic substance in a liquid such as a biological sample by utilizing an agglutination reaction (agglutination immunoassay).

[0002]

2. Description of the Related Art An immunoagglutination reaction of latex particles is caused by a specific antigen-antibody reaction with an antibody or antigen in serum using an immunoassay reagent in which an antigen or an antibody is carried on latex particles as a polymer carrier. Measurement of the antibody or antigen in serum by detecting the degree of aggregation of the polymer carrier has been conventionally performed as one of immunoserologic diagnostic methods, such as rheumatoid factor, anti-streptolysin. -O (ASO), C-reactive protein (CRP), etc.

[0003] A polyclonal antibody is usually used as an antibody in a reagent for immunoassay using latex particles. However, it is difficult to always obtain a polyclonal antibody of the same quality at the stage of production, so that a polyclonal antibody is purified into a specific antibody. The loss of the antibody during the step is also large.

[0004] As a method for solving the above problems, a monoclonal antibody can be mass-produced by using a monoclonal antibody instead of a polyclonal antibody. As a latex agglutination method using a monoclonal antibody, JP-A-6-167 describes
No. 495, an antigenic substance in a test sample is adsorbed or bound to insoluble carrier particles, and a monoclonal antibody specifically reacting with the antigenic substance is reacted, and then selectively binds to the monoclonal antibody. An agglutination immunoassay method comprising reacting a second antibody (an anti-monoclonal antibody-produced animal immunoglobulin (IgG) antibody derived from an animal other than an animal species producing a monoclonal antibody) to selectively agglutinate insoluble carrier particles. Has been proposed. However, this method has a problem that the sensitivity is low, the production of the second antibody is performed from an animal, so that the production is difficult, and it is difficult to stably obtain the antibody having the same specific activity.

[0005]

SUMMARY OF THE INVENTION In view of the above drawbacks, the present invention uses a monoclonal antibody of the same quality by a simple operation, and increases the antigenic substance in a test sample by aggregation of a polymer carrier. It is an object of the present invention to provide an agglutination immunoassay method capable of measuring at a measurement sensitivity.

[0006]

In the agglutination immunoassay according to claim 1 (hereinafter, the invention of claim 1 is referred to as the present invention 1), an antigenic substance in a test sample is adsorbed or bound to a polymer carrier. After the reaction, a monoclonal antibody specifically reacting with the antigenic substance, and protein A, a protein A-like substance that selectively binds to the monoclonal antibody,
At least one selected from the group consisting of protein G and protein G-like substance is further reacted to selectively aggregate the polymer carrier.

In the agglutination immunoassay method according to claim 2 (hereinafter, the invention according to claim 2 is referred to as the present invention 2), an antigenic substance in a test sample is adsorbed or bound to a polymer carrier, and then the test is performed. The agglutination immunoassay according to claim 1, wherein a monoclonal antibody specifically reacting with the antigenic substance is reacted without removing the test sample by washing.

The test sample used in the present invention 1 includes:
Test samples used in ordinary immunoassays can be mentioned, and preferably blood samples, plasma samples, serum samples and the like can be mentioned.

The antigenic substance used in the present invention 1 includes antigenic substances used in ordinary immunoassays, and preferably includes biological components, viruses, bacterial components, drugs and the like. Further, it is preferably glycated hemoglobin.

The polymer carrier used in the present invention 1 is an ordinary polymer carrier, for example, a natural polymer carrier such as insoluble agarose, cellulose and insoluble dextran, polystyrene and styrene-styrene sulfonic acid ester copolymer. And a synthetic polymer carrier such as vinyl acetate-acrylate copolymer and the like, and latex particles composed of a synthetic polymer are particularly preferable. The average particle size of the latex particles used varies depending on the measuring method and the measuring instrument.
The range is preferably from 0.5 to 1.0 μm.

[0011] The monoclonal antibody specifically reacting with the antigenic substance used in the present invention 1 is Koehler &
Milstein (Nature, 256, 495
497, 1975) et al. This method is known, to prepare a hybridoma-producing cell that produces a monoclonal antibody of interest by cell fusion between an antibody-producing cell of an antigenic substance and a myeloma cell, and culturing the hybridoma-producing cell in mouse ascites or the like. And a method for obtaining the desired monoclonal antibody.

The above-mentioned monoclonal antibody is used as an antibody solution. The stock solution (for example, mouse ascites) obtained by growing hybridoma-producing cells is diluted 200 to 1000 times with a buffer such as glycine buffer. Is preferred.

At least one selected from the group consisting of protein A, protein A-like substance, protein G and protein G-like substance used in the present invention 1 has at least two sites that bind to the Fc site of a monoclonal antibody. The protein A includes natural ones and those obtained by genetic recombination. Examples of protein G include natural ones and those obtained by genetic recombination. The protein A-like substance includes an oligomer of the Fc binding region of protein A, which is prepared by genetic recombination, and the Fc binding region has a trimer,
As the polymer becomes a tetramer, a pentamer or a multimer, the cohesive force of the polymer carrier increases, and the sensitivity increases. The protein G-like substance includes oligomers of the Fc binding region of protein G, which are prepared by genetic recombination, and as the Fc binding region becomes trimer, tetramer, pentamer and multimer, the higher the amount, The cohesive force of the molecular carrier increases, and the sensitivity increases.

The above protein A, protein A-like substance,
It is preferable that at least one selected from the group consisting of protein G and protein G-like substance is dissolved in a buffer such as a glycine buffer and used at a concentration of 0.01 to 1 mg / ml.

In the present invention 1, the reaction conditions between the antigenic substance and the monoclonal antibody and the reaction conditions between the monoclonal antibody and at least one selected from the group consisting of protein A, protein A-like substance, protein G and protein G-like substance are as follows. As in the normal case, a phosphate buffer, a glycine buffer, a Tris buffer, or the like is used as a reaction medium. The pH of the reaction system is preferably from 4.5 to 9. The reaction temperature is preferably from 0 to 50C, more preferably from 4 to 40C. The reaction time can be determined appropriately.

The test sample, antigenic substance, polymer carrier, protein A, protein A-like substance,
The protein G and the protein G-like substance, etc. of the present invention 1
It is the same as that used in.

The monoclonal antibody which specifically reacts with the antigenic substance used in the present invention 2 is Koehler &
Milstein (Nature, 256, 495
497, 1975), etc., which reacts with the antigenic substance adsorbed on the polymer carrier and does not react with the free antigenic substance. This monoclonal antibody is screened by immobilizing an antigenic substance to be measured on a 96-well plate, reacting the culture supernatant of hybridoma cells with the antigen substance, and further reacting with enzyme-labeled anti-mouse immunoglobulin. The reaction is carried out by a radioimmunoassay in which the culture supernatant of the hybridoma cells is reacted with the antigenic substance labeled with, and an antibody which is reacted by the ELISA and not reacted by the radioimmunoassay is obtained.

In the present invention 2, the reaction condition between the antigenic substance and the monoclonal antibody, and the reaction condition between the monoclonal antibody and at least one selected from the group consisting of protein A, protein A-like substance, protein G and protein G-like substance Is the same as in the first invention.

In the measurement method of the present invention 1, first, the polymer carrier is added to a test sample, and the antigenic substance in the sample is adsorbed or bound to the polymer carrier to fix the antigenic substance. Phase. Next, at least one selected from the group consisting of the monoclonal antibody and protein A, protein A-like substance, protein G and protein G-like substance is added to the sample containing the solid-phased antigenic substance, Reaction between the immobilized antigenic substance and the monoclonal antibody,
And causing a reaction between the monoclonal antibody and at least one selected from the group consisting of protein A, protein A-like substance, protein G and protein G-like substance, whereby the immobilized antigenic substance-monoclonal antibody-protein A, protein A Aggregation of the polymer carrier is caused by forming a complex having a structure of at least one member selected from the group consisting of a like substance, protein G, and a protein G-like substance, a monoclonal antibody, and an immobilized antigen substance. Next, the degree of the aggregation is optically measured based on, for example, the intensity of scattered light, the absorbance, and the intensity of transmitted light. The wavelength for the measurement is preferably 300 to 2400 nm, and it is preferable to use an automatic analyzer as the measuring device.

The solidification of the antigenic substance by adsorbing or binding the antigenic substance in the sample to the polymer carrier is preferably performed in a buffer to prevent self-aggregation of the polymer carrier.

When adding the above-mentioned monoclonal antibody and at least one selected from the group consisting of protein A, protein A-like substance, protein G and protein G-like substance to a sample containing an immobilized antigenic substance, , A monoclonal antibody and at least one selected from the group consisting of protein A, protein A-like substance, protein G and protein G-like substance may be added separately, but in order to reduce non-specific reaction, Antibodies and protein A, protein A-like substances,
It is preferable to add a substance in which at least one selected from the group consisting of protein G and protein G-like substance is combined.

The assay method of the present invention uses a monoclonal antibody which reacts with an antigenic substance to be measured adsorbed on a polymer carrier and does not react with a free antigenic substance. Other than reacting a monoclonal antibody specifically reacting with the antigenic substance without adsorbing or binding the antigenic substance therein to the polymer carrier without removing the test sample by washing, the present invention It is the same as the measurement method of 1.

[0023]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The embodiments of the present invention will be specifically described below by taking the measurement of glycohemoglobin A1c (HbA1c) as an example.

Reference Example 1 Acquisition of HbA1c and HbA0 (HbA0 is non-glucosylated hemoglobin) Human blood was centrifuged at 2500 rpm for 20 minutes to remove plasma proteins. Physiological saline was added and centrifugal washing was performed twice. Next, an equal amount of distilled water was added to the centrifuged red blood cell pellet to cause hemolysis. Next, high-speed centrifugation was performed at 15000 rpm for 45 minutes to remove precipitates, and the supernatant was purified using a BioRex70 column (manufactured by BioRad) according to the method of Trivelli et al. (New Engl. J. Med. 35
3 (1971)) to fractionate HbA1c and HbA0.

Reference Example 2 Preparation of Monoclonal Antibody Against the N-terminal Glycopeptide Epitope of HbA1c β Chain The following two peptides corresponding to the N-terminal amino acid sequence of hemoglobin β chain were synthesized using a peptide synthesizer (Applid B).
The product was synthesized using iosystems (Model 430A). 1) Val (valine) -His (histidine) -Leu
(Leucine) -Thr (threonine) -Pro (proline) -Cys (cysteine) 2) Val (valine) -His (histidine) -Leu
(Leucine) -Cys (cysteine)

The crude peptide obtained by the above synthesis was
g / ml in pure water and cooled to 4 ° C.
A 1.5-fold molar ratio of 2,2'-dithiodipyridine was added dropwise with stirring, and the mixture was reacted for 10 minutes to obtain a cysteine S
The H group was protected. After lyophilization, it was dissolved in 1% acetic acid, and the insoluble matter was removed by centrifugation. Then, Sephadex G25 (15 ×
The peptide fraction obtained by gel filtration with a 900 mm column) was lyophilized. This peptide is dissolved in acetic acid, glucose is added at twice the molar ratio in the presence of pyridine,
Stirred for 0 days. The solution after stirring was subjected to high performance liquid chromatography (hereinafter, referred to as HPLC) under the following separation conditions to obtain a glycopeptide, which was lyophilized.

Separation conditions Column: TSKgel, ODS-120A (21.5 ×
Mobile phase: gradient from 10% acetonitrile / 0.1% trifluoroacetic acid to 60% acetonitrile / 0.1% trifluoroacetic acid Flow rate: 5 ml / min Monitor: absorbance 280 nm

The lyophilized peptide was dissolved in a 0.1 M potassium phosphate buffer (pH 8.5), and a three-fold amount of D
Addition of isothreitol (manufactured by Wako Pure Chemical Industries, Ltd.), reaction with nitrogen gas for 24 hours, elimination of the cysteine protecting group, adjustment of pH to 5 with hydrochloric acid, and change of absorbance to 215 nm under the above HPLC separation conditions A glycopeptide was obtained.

N-succinimidyl 6-malemid was used as a spacer for this glycopeptide.
Using ocaproate (manufactured by Wako Pure Chemical Industries, Ltd.), a carrier protein (a water-soluble protein such as Thyroglo
Bulin was bound to Edestin (both manufactured by Fluka) as an insoluble protein to obtain an immunogen.

100 μg of the above immunogen was made into micelles with Freund's complete adjuvant (manufactured by Difco), and Bal
b / c mice (8 weeks old) were injected intraperitoneally. The booster boosts the immunogen with Freund's incomplete adjuvant (Difco
Micelles) and performed twice at one-month intervals. Three days before cell fusion, the immunogen in saline was injected subcutaneously and used as a booster.

Spleen cells were obtained from the spleen of the immunized mouse, mixed with mouse myeloma cell line PAI (Cancer Research Source Bank (JCRB)) at a ratio of 10: 1, and fused with polyethylene glycol 4000. Was. The cells were suspended in HAT medium, spread on a 96-well plate, and waited for clonal expansion of the fused cells.

To screen the desired monoclonal antibody-producing hybridoma, a 96-well ELISA plate (NUNC-IMMUNOPLATE, MAXISO) preliminarily immobilized with HbA1c (obtained in Reference Example 1) was used.
A 96-well ELISA plate (material name is the same as described above) on which RPF96 (4-42404)) and HbA0 (obtained in Reference Example 1) were immobilized was used. The production of this immobilized plate is performed by adding HbA1c to each well of the plate.
50 mM citrate buffer dissolved at a concentration of μg / ml (p
H5.6) was dispensed in 50 μl aliquots.
And left overnight. In addition, the above HbA
HbA0 was similarly used except that 1c was replaced with HbA0.
A solid-phased plate was prepared.

The screening method is specifically described below. First, the above HbA1c-immobilized plate and HbA
After washing the immobilized plate four times with PBS-T (0.01 M phosphate buffer, pH 6.8 / 0.15 M NaCl / 0.1% Tween 20), culture in which the above-mentioned fused cells have undergone clonal expansion. 50 μl of the supernatant was added, and left at room temperature for 2 hours to react. After washing 4 times with PBS-T, 50 μl of peroxidase-labeled anti-mouse IgG (manufactured by CAPPL) diluted 2500-fold with PBS-T was used.
l was added and reacted at room temperature for 1 hour. After washing with PBS-T four times, 0.1% o-phenylenediamine / phosphate-citrate buffer (citric acid monohydrate 7.3 g, disodium phosphate dodecahydrate 23.9 g, pure water 1000 ml / 0.
(003% H ₂ O ₂ ) was added, and the mixture was reacted at room temperature for 30 minutes. Then, 50 μl of 2N sulfuric acid was added to stop the enzyme reaction. Next, the absorbance at 490 nm of the solution after the enzyme reaction was measured using an immunoplate reader (manufactured by Dynatech, MR5000).

According to the above screening method, HbA1c
A hybridoma producing a monoclonal antibody that did not react with HbA0 was selected and cloned.

After culturing the obtained clones in large quantities, one week
Immediately before, 1 ml of pristane (Sigma) was injected intraperitoneally.
Balb / c mice (9 weeks old) were hybridoma 1 ×
10 ⁷cells were injected intraperitoneally with saline,
When the water has filled the abdominal cavity, the monoclonal antibody is
Collected as ascites.

Reference Example 3 Preparation of Test Sample Diluent Bovine serum albumin was added to 50 mM HEPES buffer (pH 6.5) (manufactured by Wako Pure Chemical Industries, Ltd.) at 0.25% (W /
V), polyethylene glycol 6000 (PEG600
0, average molecular weight 7500, manufactured by Nacalai Tesque).
The sample was dissolved so as to have a concentration of 5% (W / V) to prepare a test sample diluent.

Example 1 50 μm of whole blood collected from a human
1 ml of distilled water was added to the mixture to cause hemolysis, thereby preparing a hemolysis stock solution. Further, a glycine buffer solution (hereinafter referred to as GBS 0.0
5M glycine, pH 8.2 / 0.15M NaCl /
8 times, 16 times, 32 times and 64 times with 0.02% NaN ₃ ).
The test sample was diluted by a factor of two.

The test sample 1 was placed in a polystyrene test tube.
Then, a polystyrene latex having a particle size of 0.087 μm (manufactured by Sekisui Chemical Co., Ltd.) was diluted with GBS to give a latex suspension having a concentration of 0.15% by weight.
HbA1c in the test sample was adsorbed to the latex (this was HbA1c).
A1c-latex adsorbent).

The anti-HbA1c monoclonal antibody obtained in Reference Example 2 was diluted 600-fold with GBS containing 0.3% Tween 20 (hereinafter referred to as GBS-T).
00 μl and 1 mg of protein A (manufactured by Wako Pure Chemical Industries)
25 μl of the solution dissolved in 10 ml of BS-T was added to 37
The reaction was carried out at 20 ° C. for 20 minutes to prepare an anti-HbA1c monoclonal antibody-protein A conjugate (hereinafter, referred to as an antibody reaction solution).

Using an automatic biochemical analyzer (Hitachi 7050, manufactured by Hitachi, Ltd.), 350 μl of the test sample diluent prepared in Reference Example 3 and 350 μl of the H
The bA1c-latex adsorbed solution was added at a rate of 50 μl and measured. The measurement temperature was 37 ° C. The reaction amount was HbA
The measurement was performed by measuring the change in absorbance (measurement wavelength: 570 nm) between 80 seconds and 320 seconds after the addition of the 1c-latex adsorbent. Table 1 shows the obtained change in absorbance.

Example 2 The procedure of Example 1 was repeated except that the protein A (manufactured by Wako Pure Chemical Industries) was replaced by a recombinant protein A having an IgG binding region (manufactured by Sigma). Operated. Table 1 shows the obtained change in absorbance.

(Comparative Example 1) Rabbit anti-mouse Ig in place of protein A (manufactured by Wako Pure Chemical Industries, Ltd.) in Example 1.
The same operation as in Example 1 was performed except that G antibody (manufactured by Kappel) was used. Table 1 shows the obtained change in absorbance.

[0043]

[Table 1]

[0044]

The composition of the agglutination immunoassay of the present invention 1 is as described above. After the antigenic substance in the test sample is adsorbed or bound to the polymer carrier, the same quality can be used for a simple operation. And a monoclonal antibody that specifically reacts with the antigenic substance, and at least one selected from the group consisting of protein A, protein A-like substance, protein G, and protein G-like substance that selectively bind to the monoclonal antibody. The present invention provides an agglutination immunoassay method in which a polymer carrier is selectively agglutinated by further reacting the same, so that measurement can be performed with high measurement sensitivity. The configuration of the agglutination immunoassay of the present invention 2 is as described above. After the antigenic substance in the test sample is adsorbed or bound to the polymer carrier, the antigen is removed without removing the test sample by washing. The agglutination immunoassay according to claim 1, wherein the agglutination immunoassay is performed by reacting a monoclonal antibody that specifically reacts with an aggressive substance. Therefore, an agglutination immunoassay that can be measured more easily and with high measurement sensitivity is provided.

Claims (2)

[Claims] 1. A monoclonal antibody which specifically reacts with an antigenic substance in a test sample after adsorbing or binding to the polymer carrier, and a protein A which selectively binds to the monoclonal antibody. An aggregation immunoassay method, wherein at least one selected from the group consisting of protein A-like substance, protein G and protein G-like substance is further reacted to selectively aggregate polymer carriers. 2. After adsorbing or binding the antigenic substance in the test sample to the polymer carrier, the monoclonal antibody specifically reacting with the antigenic substance is reacted without removing the test sample by washing. The agglutination immunoassay according to claim 1, which is performed.