JPH03150465A - Reagent for measuring activated platelet antigen - Google Patents

Abstract

PURPOSE:To obtain the measuring reagent which immunologically measures the quantity of GMP 140 in a sample, such as bodily fluid, by using >=2 kinds of antibodies which recognize the same GMP 140 but vary in antigen recognizing sections as constituting components. CONSTITUTION:The GMP 140 to be used as the antigen for the antibody is formed by solubilizing the membrane protein of the platelet prepd. from the fresh human blood by a centrifugal sepn. method and washing method by a surfactant, etc., and by refining the GMP 140 by affinity chromatography, etc., and is used as the antigen at the time of preparing the antibody. The antiserum to the GMP 140 is prepd. by an already established method. The quantity of the GMP 140 in the sample is measured by using the carrier conjugated with the anti-GMP 140 specific antibody (insoluble antibody) and the enzyme-labeled anti-GMP 140 specific antibody (labeled antibody). These anti-GMP 140 antibodies are used as the reagent for measuring the GMP 140.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel reagent for measuring GMP 140, and more specifically to a method for measuring GMP 140 using an enzyme immunoassay.
140 measurement reagents.

[Conventional technology]

In the human body, when the vascular endothelium is injured and the endothelial tissue is exposed, platelets in the bloodstream adhere to this site and release various platelet-stimulating substances.

These substances activate new platelets, which aggregate with previously adhered platelets to form a thrombus. The formation of thrombi causes myocardial infarction, cerebral thrombosis, etc., and the progression of arteriosclerosis. The state in which platelets are activated in this way can be regarded as a state of thrombotic readiness or thrombotic tendency, and prompt diagnosis of this state is clinically important in understanding the pathology of thrombosis. It has been desired to establish a quick and simple diagnostic method because of its significance and from the standpoint of emergency testing.

To date, diagnosis of platelet activation has mainly been made by measuring plasma concentrations of β-thromboglobulin, platelet factor 4, thromboxane B2, etc., but all of these proteins have short half-lives in the blood. The shortcoming was that it was extremely short, making it difficult to monitor transient increases in platelet activation.
29, No. 13, p. 1334 (1984)].

Furthermore, a glycoprotein with a molecular weight of 140,000 that specifically appears on the membrane surface of activated platelets and does not appear on resting platelets has recently been identified, and a platelet-containing sample is brought into contact with a radioactively labeled monoclonal antibody for the protein. A method for assaying activated platelets in a platelet-containing sample was developed in which labeled immune complexes were measured as a measure of activated platelets in the sample [JP-A-61-192285].

This substance is called GMP 140 or PADGEM glycoprotein.

In addition, a competitive RIA method was developed using a polyclonal antibody specific for GMP 140 [Berman C,L, et al., Methods in Enzymology].
nzymology) Volume 169, Page 321 (198
9)]. This method uses pre-radioactively labeled GM.
P140 antigen and sample competitively with one anti-GMP14
0 rabbit antibody, reacted overnight at 4°C, reacted with anti-heron TgG antibody, added polyethylene glycol to generate an immunoprecipitate, and measured the radioactivity of the precipitate. This is to measure the amount of GMP140 in the sample.

[Problem to be solved by the invention]

The above-mentioned radioimmunoassay requires at least one day of operation time, is not suitable for emergency testing, and has problems in terms of complicated operation, environmental contamination with radioactive substances, and effects on the human body. When antiserum is used as an antibody, there is also a problem in the specificity of its reactivity. In addition, this antiserum and radiolabeled GMP14
In order to prepare GMP140, a large amount of GMP140 had to be purified, which was also a big problem.

The present invention has been made to overcome the problems of the conventional measuring methods described above, and its purpose is to provide a measuring reagent for immunologically measuring the amount of GMP 140 in samples such as body fluids. be.

[Means to solve the problem]

If we go beyond the present invention, the present invention is characterized in that a reagent for measuring GMP140 in a sample is composed of two or more types of antibodies that recognize the same GMP140 but have different antigen recognition sites. The present invention relates to a GMP 140 measurement reagent.

The present invention enables faster, simpler, and safer GMP than conventional methods 1
It became possible to mass produce 40 measurement reagents.

When preparing the antibody, GMP140 is used as an antigen by solubilizing platelet membrane protein prepared from fresh human blood by centrifugation and washing with a surfactant, and then using affinity chromatography to obtain GMP140.
40 can be purified and used as an antigen. GMP
Antiserum against 140 can be prepared by established methods.

That is, an experimental animal such as a rabbit may be immunized with GMP 140 several times, and serum may be extracted from the animal after an appropriate period of time. Enzyme immunoassay (ELIS)
A) The presence of specific antibodies in the antiserum can be tested by techniques such as Western blotting.

In order to obtain specific antibodies using this method, the antigen used for immunization must be a single protein with high purity. Furthermore, in order to immunize large experimental animals such as rabbits and goats, it is necessary to prepare a large amount of antigen.

On the other hand, for the preparation of anti-GMP 140 antibody, the method of Kohler and Milstein was used.
.

Neci + - (Nature), Volume 256, No. 47
6 (1975)], there is no need to prepare a large amount of antigen, and there is no need to purify the antigen to absolute purity. This is because the lung cells of an experimental animal immunized with an antigen are fused with myeloma cells, and cloned cells that produce specific antibodies are selected under in vitro conditions.

In particular, mice are often used as experimental animals.

For example, GMP 140 was added intraperitoneally to Ba1b/C7 mice.
Alternatively, the washed platelets themselves can be used for immunization, followed by a booster immunization 4 weeks later, and 3 days later, the pancreas is removed from the mouse. Lung cells and mouse myeloma cells are fused by the action of polyethylene glycol and cultured in a 96-well plate using conventional methods. Collect the supernatant of each culture and ET
-1SA method to select cells producing specific antibodies, further cloning by limiting dilution method, anti-GM
Obtain P1.40 monoclonal antibody producing cells (hybridoma). These hybridomas secrete monoclonal antibodies in the culture medium.

Hybridomas can be cultured using a commercially available serum-free medium, and an antibody fraction can be obtained from the supernatant by operations such as ammonium sulfate weir separation. Alternatively, a hybridoma can be cultured in a mouse of the same type, and an antibody fraction can be obtained from the animal's body fluid by the same procedure.

Measurement methods using two or more antibodies with different antigen specificities for GMP 140 include immunoassay methods well known in this field, such as enzyme immunoassay,
Radioimmunoassay, immunoturbidimetry, latex agglutination method, hemagglutination method, 5RID method (immunodiffusion method), etc. are used. Among them, enzyme immunoassay is the most practical in terms of speed, sensitivity, simplicity, etc. i.e. anti-GMP 14
The anti-GMP140-specific antibody is treated with an insoluble carrier such as polystyrene beads, glass beads, or a polystyrene microtiter plate, and covalently bonded or physically adsorbed to these carriers to obtain an insoluble carrier bound with the anti-GMP140-specific antibody. On the other hand, an anti-GMP 140-specific antibody having a different specificity is labeled with an enzyme using a conventionally known method. for example,
A compound optimal for the enzyme used (eg, m-maleimide ester for β-galactosidase, periodic acid for peroxidase) and then an antibody are coupled to this reaction to yield an enzyme-labeled anti-GMP 140-specific antibody. The thus obtained anti-GMP 140-specific antibody-bound carrier (insoluble antibody) and the enzyme-labeled anti-GMP 140-specific antibody (
The amount of GMP 140 in a sample can be measured using labeled antibodies), and these anti-GMP 140 antibodies are used in the GMP 140 measurement reagent of the present invention.

From the viewpoint of specificity and rapidity, it is essential to use two or more types of anti-GMP 140 antibodies with different specificities for use in these measurement reagents. The antibody used may be either an antiserum and/or a monoclonal antibody, but monoclonal antibodies are suitable.

This is because the use of monoclonal antibodies increases specificity and allows a very wide range of concentrations of GMP 140 to be measured. Furthermore, as the antibody species used, IgG is particularly superior in terms of ease of labeling.

For example, two different types of anti-GMP 140 monoclonal antibodies can be used as antibodies for use in antibody solid phase preparations or enzyme-labeled antibodies.

In this case, both are directed against GMP 140, but in particular those directed against distinct epitopes of GMP 140 are used.

Furthermore, these antibodies are not limited to being used as immunoglobulins; F(ab') 2. which can be obtained by treating these antibodies with pepsin. It can also be used as a fragment such as Fab'. When these fragments are used, non-specific adsorption can be prevented.

An example of a measurement using the reagent for measuring GMP 140 according to the present invention is as follows: a) A sample is simultaneously treated with an anti-GMP 140 antibody or antibody fragment-bound solid phase and an enzyme-labeled anti-GMP 140 antibody or antibody fragment in one step. G in the sample
Forming a sandwich between MP 140, an immobilized antibody or antibody fragment, and an enzyme-labeled antibody or antibody fragment; b) Measuring enzyme activity by reacting the enzyme on the resulting sandwich with a substrate specific to the enzyme. It consists of doing.

As the insolubilizing carrier used in the antibody-binding solid phase material, those having the ability to bind antibodies can be used, such as polystyrene, polypropylene, polyvinyl chloride,
Alternatively, polycarbonate microplates, beads, sticks, test tubes, etc. may be used.

Enzyme-labeled antibodies can also be produced by enzyme-labeling using methods well known to those skilled in the art, such as the maleimide method and the 0-pyridyl disulfide method. Examples of enzymes used for enzyme labeling include alkaline phosphatase, β-
Examples include D-galactosidase, glucose oxidase, acetylcholinesterase, lactate dehydrogenase, glucoamylase, tyrosinase, horseradish peroxidase, lactoperoxidase, and horseradish peroxidase is preferred.

The substrate may be any substrate specific to the enzyme used for enzyme labeling. For example, when the enzyme is horseradish heloxidase, 0-phenylenediamine may be used as a substrate, and the color generated by the enzyme reaction may be measured as absorbance at a wavelength of 492 nm. The amount of GMP 140 can be calculated and measured from the enzyme activity thus determined.

As described above, the present invention provides a new and simple measuring reagent for measuring GMP 140 in samples, and is useful for predicting the onset of myocardial infarction and cerebral thrombosis, determining the application of antithrombotic drugs, and for thrombosis after vascular bypass surgery. diagnosis of trends,
It is used in emergency diagnosis.

〔Example〕

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

Example 1 (1) Preparation of washed platelets 700 g of blood collected from a normal person with addition of citrate
After obtaining the platelet fraction by centrifugation for 1 minute, RCD
Solution (36mM citric acid, 5mM glucose, 1mM
MgCl+, 5mM KCl, 103mM NaC
The platelets, which had been centrifugally washed twice with 20 ng/mf protein and 20 ng/mf protein, were further washed with albumin density gradient
The platelets were washed using the ENT method and resuspended in Ca2+-free HEPE 5-Tyrade buffer to prepare washed platelets.

(2) To the washed platelets obtained in step (1), crude purification of GMP140 antigen, Triton Embryo agglutinin (whea)
t germ agglutinin) was passed through an immobilized agarose column, and both components adsorbed on the column were eluted with 0.1M glycine-1101 buffer (pH 2.2),
Collected as crude GMP 140 antigen.

(3) Preparation of antiserum against GMP140 Crude GMP
140 antigen (0.5 mg) was dissolved in 0.57 mg of physiological saline, and an equal volume of complete Freund's adjuvant was added to this.
After emulsification, it was injected subcutaneously into rabbits. The same amount of antigen emulsified with incomplete Freund's adjuvant was subcutaneously injected four times every two weeks, and whole blood was collected 10 days after the final immunization, left at room temperature for 60 minutes, and then centrifuged. An antiserum containing anti-GMP 140 antibody was obtained by doing this. The specificity and titer of the third antibody were confirmed by methods well known to those skilled in the art, ie, ELISA and Western blotting.

(4) Preparation of monoclonal antibody against GMP 140 by platelet immunization method 2 x 10 8 freshly washed platelets were mixed with 0.0 ml of physiological saline. fml
An equal volume of complete Freund's adjuvant was added to emulsify the mixture, and the mixture was injected intraperitoneally into Balb/c mice. Four weeks later, 2×10" washed platelets were intraperitoneally injected into the mice. Three days later, lung cells were obtained from the lungs removed from the mice, and mouse myeloma cells (SP210) were obtained.
and cell number at a ratio of 10:1 in the presence of 50% polyethylene glycol and 20% dimethyl sulfoxide.
Cell fusion was performed by leaving it for a minute. After diluting by adding serum-free DMEM medium, remove the supernatant by centrifugation, and
Cells were suspended in DMEM medium containing 0% tallow serum,
The cells were dispensed into a 6-well microtiter plate at 2×10' cells per well. After that, half of the medium was replaced with HAT medium every 1 to 3 days, and after 10 to 20 days, the culture supernatant of the well in which the fused cells (hybridoma) had grown was collected, and the presence or absence of antibody production was determined by ELTSA method etc. Research, GM
Three hybridoma strains producing antibodies against P140 were selected.

These hybridomas were cloned twice by the limiting dilution method, and one clone, PL7-6, was obtained as the hybridoma clone that produced the highest antibody titer.

It was confirmed by Western blotting that the monoclonal antibodies produced by these hybridomas specifically reacted with GMP 140. In order to obtain large quantities of these monoclonal antibodies, approximately 2 x 10' hybridomas were intraperitoneally injected into Ba1b/c mice to form ascites tumors. After 10 days, ascites was collected, and ammonium sulfate was extracted from the ascites using a conventional method. Anti-GMP 14 by analysis and DEA5 E cellulose chromatography
0 monoclonal antibody, a pH 7-6 monoclonal antibody, was purified. It was confirmed by EIA that the pH7-6 monoclonal antibody was of the IgG+ class. This loan strain was named hybridoma PL'l-6, and H
It is designated as P. hybridoma PL 7-6 and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P-11073.

(5) Preparation of antibody-bound beads The anti-GMP 140 antiserum obtained in (3) above was subjected to ammonium sulfate salting out according to a conventional method, and the anti-GMP 1.40 antibody was purified by DEAE cellulose chromatography. Antibody 1mg
0, I M U acid buffer y (pi
(8,0>20m1. Add 100 polystyrene balls (manufactured by Tanezu Kagaku Co., Ltd., particle size 6.35 mm), and heat at 5°C.
The antibody was immobilized on the beads by reacting for 6 hours and 1 hour at 37°C. After washing the beads thoroughly with physiological saline, they were immersed in 10 mM phosphate buffer (pH 7.4) containing 1% 6 bovine serum albumin (BSA), 0.05% sodium azide, 0.9% NaC, and Leave overnight at °C and remove anti-G
MP 140 antibody-conjugated beads were obtained.

(6) Preparation of monoclonal antibody enzyme labeled product (
Anti-GMP 140 monoclonal antibody 1 obtained in 4)
) Peroxidase (manufactured by Boehringer-Mannheim) was added to L7-6 using the method of Nakane et al.
Journal of Histochemistry and Cytochemistry (J, formerly stochem, Cytochem
), Vol. 22, p. 1084 (1974)) to obtain a labeled antibody. That is, 10 mg of peroxidase is dissolved in 2 scoops of purified water, and 0.2 mm of 0.1 M potassium periodate is added. After reacting at room temperature for 20 minutes, the mixture was dialyzed against 1 mM acetate buffer (pH 4,0) at 4°C overnight. Add to this 0.2M carbonate buffer (pH 9,
Add 5) and adjust the pH to 9-9.5. On the other hand, anti-GM
2 mg of P7 140 monoclonal antibody was dissolved in 1.5-phosphate buffered saline (pH 7.4) and dialyzed against 10 mM carbonate buffer (pH 9.5) overnight at 4°C. This was mixed with the above peroxidase treated with periodic acid and reacted at room temperature for 2 hours, and then sodium borohydride (4 mg/g) was added at 0.1 if! After reaction at 4°C for 2 hours, phosphate buffered saline (pil
cA22 (LKB
The fraction was fractionated by gel filtration. Collect the portions with matching peroxidase activity and antibody activity, add sodium merthiolate to a final concentration of 0.01%, and
Stored at °C.

(7) Measurement of GMP140 The EIA method was performed as follows. Put 200 μl of the sample into a tube, and add 1 insolubilized antibody beads into the tube.
A first incubation of 20 molecules (1 Jll) is performed at 37°C. Next, the beads are washed three times with three volumes of physiological saline, and 200 μp of the 8-labeled antibody solution (300-fold dilution) is added to the tube containing the beads, and a second incubation is performed at 37° C. for 20 minutes. Next, wash the beads three times with 3 ml of saline, transfer the beads to another tube, and add color reagent 3 to this.
00μII (o-phenylenediamine 1mg/m, H
0.01% of 2O2, 0゜1M citrate buffer pH
5.0) was added thereto, and the mixture was reacted at 37°C for 10 minutes, and 1 ml of IN H2SO, was added to stop the reaction. Absorbance at a wavelength of 492 nm was measured.

Note that FIG. 1 is a graph showing the calibration curve used in the measurement reagent in Example 1 of the present invention as a relationship between GMP 140 concentration (ng/mf, horizontal axis) and absorbance at 492 nm (vertical axis).

Example 2 (1) Preparation of monoclonal antibody against GMP 140 by soluble antigen immunization method 50μ of GMP 140 antigen obtained in Example 1-(2)
Dissolve g in 0.1 mR of physiological saline, add an equal amount of 9 ml complete Freund's adjuvant, emulsify, and make Ba1
b/c mice were injected intraperitoneally. Antigen 50 after 4 weeks
μ8 alone was injected intraperitoneally into the same mice. From the lungs removed from the mouse 3 days later! 111! Obtain visceral cells,
Mouse myeloma cells (SP210) and cell number 10 = 1
50% polyethylene glycol and 20%
% dimethyl sulfoxide for 1 minute to perform cell fusion. Serum-free ■] After diluting by adding MEM medium, remove the supernatant by centrifugation, suspend the cells in DMEM medium containing 10% tallow serum, and put 2 x 10' cells per hole in a 96-well microtiter plate. It was dispensed as follows. Afterwards, add half of the medium to HAT every 1 to 3 days.
After 10 to 20 days, the culture supernatant of the well in which the fused cells (hybridoma) had grown was collected, and the presence or absence of antibody production was examined by ELISA, etc., and GMP 14
Three hybridoma strains producing antibodies against 0 were selected.

0 These hybridomas were cloned twice by the limiting dilution method, and the clone strain WGA-1 was selected as the hybridoma clone that produced the highest antibody titer.
acquired one share of

It was confirmed by Western blotting that the monoclonal antibodies produced by these hybridomas specifically reacted with GMP 140. In order to obtain large quantities of these monoclonal antibodies, approximately 2 x 107 hybridomas were intraperitoneally injected into Ba1b/c mice to form ascites tumors, and the ascites was collected 10 days later and subjected to salting out with ammonium sulfate using DEAE. Anti-GMP140 monoclonal antibody, i.e., W
GΔ1 monoclonal antibody was purified.

WGA, -1 monoclonal antibody was converted to Ig by EIA method.
I confirmed that it was a G+ class.

This loan strain was named hybridoma WGA-1, and H
It is designated as Ybridoma W G A-1 and is designated as F.E.
> has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

In addition, WGA-1 monoclonal antibody and P
It was confirmed by competitive EIA that the L7-6 monoclonal antibody has a different antigen recognition site from GMP 140.

(2) Preparation of monoclonal antibody-conjugated beads (
Anti-GMP 140 monoclonal antibody WGA obtained in 1)
0, 1M phosphate buffer containing 1 mg of -1 (
pH 8,0) Add 100 polystyrene balls (manufactured by Tanesui Kagaku Co., Ltd., particle size 6.35 mm) to 20 bottles, and incubate at 5°C.
The antibody was immobilized on the beads by reacting for 6 hours and 1 hour at 37°C. After thoroughly washing the beads with physiological saline, they were immersed in 10 mM phosphate buffer (p17.4) containing 1% bovine serum albumin (BSA), 0.05% sodium azide, and 0.9% NaCl. Leave overnight at °C to remove anti-GM
P140 monoclonal antibody-coupled beads were obtained.

2 (3) GMP measurement EIA method was performed as follows. Sample 20μl
into a tube, and then add P I = 7- as described in Example 1.
Add 200 μp of 6-labeled antibody solution (300-fold dilution) and place insolubilized antibody beads one by one into tubes and incubate at 37°C for 3
Perform the first incubation for 0 minutes.

The beads were then washed three times with 3 ml of saline, the beads were transferred to another tube, and this was added with 300 μf of color reagent.
l (o-phenylenediamine 1. mg/ml,
0.01% of H2O2, 0. IM citrate buffer p
l+5.0) was added and reacted at 137°C for 10 minutes to give I N l128[).

1mf! was added to stop the reaction. Wavelength 492nm
The absorbance was measured.

Note that FIG. 2 is a graph showing the calibration curve used in the measurement reagent in Example 2 of the present invention in terms of the relationship between GMP 140 concentration (ng/mf, horizontal axis) and absorbance at 4920 m (vertical axis).

〔Effect of the invention〕

3. A quick and simple reagent for measuring GMP 140 in biological samples has been provided.

[Brief explanation of the drawing]

FIG. 1 is a graph showing a calibration curve used in the measurement reagent in Example 1 of the present invention, and FIG. 2 is a graph showing a calibration curve used in the measurement reagent in Example 2 of the invention.

Claims (1)

[Claims] 1. A GM characterized in that a reagent for measuring GMP140 in a sample is composed of two or more types of antibodies that recognize the same GMP140 but have different antigen recognition sites.
P140 measurement reagent.