JPH03287070A - Method for measuring fibrinopeptide bbeta1-42 - Google Patents

Abstract

PURPOSE:To measure many specimens with a good sensitivity by bringing the same into contact with the labeled antibodies which specifically recognize immune reaction products, removing the free labeled antibodies and directly and indirectly detecting the labels. CONSTITUTION:The anti-FPBbeta1-42 antibodies are refined by a method used for refining ordinary proteins from the monoclonal antibodies produced by hybridomas. The antibodies are immobilized to a solid phase. The labeled antibodies which specifically recognize the unlabeled anti-FPBbeta1-42 antibodies or the immune reaction products thereof are used and the FPBbeta1-42 in the sample is measured by a sandwich measurement when these antibodies are used. There are no particular limitations on order to bring the immobilized antibodies, the labeled antibodies and the sample into contact with each other. The formed immune reaction products are separated from the free labeled antibodies and the labels are directly or indirectly detected. The concn. of the FPBbeta1-42 in the sample is measured in a 0.1 to 200ng/ml range in this way.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for measuring FPBβ, -42 in a sample using a monoclonal antibody that specifically recognizes FPBβ, -42.

(Prior art) It has been pointed out that in patients with thrombosis or a condition that is prone to thrombosis, the blood is in a so-called hypercoagulable state, and the hypercoagulable state is caused by activation of the vasotropic coagulation system due to some cause. It is believed that.

Blood coagulation progresses through a cascade reaction, which includes the fibrinogen-fibrin system, and an increase in the activity of enzymes such as thrombin involved in the decomposition of fibrinogen means activation of the vasotropic coagulation system.

Fibrinogen is a glycoprotein with a molecular weight of about 340,000, and has the form of a dimer consisting of a total of six pairs of Aα, Bβ, and γ polypeptide chains connected by S-8 bonds. When the blood coagulation system is activated, fibrinogen is converted to fibrin by thrombin, and this process is a central reaction located at the final stage of the blood coagulation process. First, when thrombin acts on fibrinogen, it cleaves between ArglB and c+yi7 near the N-terminus of the Aα chain, producing fibrinopeptide A (FPA) called Alal-ArglB. The FPA released becomes a desfibrin monomer, and the part responsible for binding fibrin is exposed. Furthermore, it is converted to fibrin by the action of thrombin, but the region between rg14 and Gly15 near the N-terminus of the Bβ chain is cleaved, resulting in fibrinopeptide B (FPB) consisting of 14 amino acids.
) is released, thereby exposing the parts involved in fibrin binding. Fibrin is polymerized from a monomer to a polymer, and then becomes stabilized fibrin through crosslinking by activated factor X.

On the other hand, plasmin produced by activation of the fibrinolytic system is
When plasmin acts on fibrinogen or desA fibrin monomer to cleave the Arg42-AIa43 bond on the MN terminal side, fibrinopeptide B1-42 is generated from the Bβ chain, and after releasing FPB, plasmin acts on fibrin. Then, fibrinopeptide Bβ1. ．． 42 will be released. F P
B B ,42 and FPBβ15-42 are both peptides released by plasmin, so FPB
β, -42 indicates primary fibrinolysis, FPBβ1. -42 reflects secondary fibrinolysis.

At present, FPBβ1-42 is determined by radioimmunoassay (RI).
A) It is measured by enzyme immunoassay (EIA), both of which are a type of measurement method called a competitive method. However, RIA not only requires great care in handling the reagents used therein, but also requires special facilities. Furthermore, EIA under competition law has the disadvantage of relatively low sensitivity.

(Problems to be Solved by the Invention) As described above, the conventional methods have various problems, and the purpose of the present invention is to solve these problems and provide a simple measurement method. That is, the aim is to establish a sandwich system for FPBβ1-42, a peptide of 42 amino acid residues, which has been considered difficult, and to solve the above problems without impairing detection sensitivity.

(Means for Solving the Problems) In order to solve the problems of the prior art, the present inventors have intensively studied a method for measuring FPBβ, -42 that is easy to operate and has high measurement accuracy. They found a solution to the problem and completed the present invention. That is, the present invention provides a method for measuring fibrinopeptide Bβ1-42 in a sample, comprising: (a) Fibrinopeptide Bβ1-42;
A monoclonal antibody/sample immobilized on a solid phase that specifically recognizes β1-42 and a labeled antibody that specifically recognizes fibrinopeptide Bβ1-42 at a site different from that of the immobilized monoclonal antibody. (b) If an unlabeled monoclonal antibody is used in (a), specifically recognizes the monoclonal antibody or the immune reaction product generated in a. (c) removing free labeled antibody; and (1) directly or indirectly detecting the label to quantify the immune reaction product. The present invention provides a characteristic method for measuring fibrinopeptide Bβ1-42 in a sample, and the details thereof will be explained below.

The present invention provides, on the antigen FPBβ, -42,
FPBβ1 was detected by sandwich immunoassay using two types of monoclonal antibodies with different recognition sites.
This is a method for measuring FPBβ, -42, which is characterized by quantifying -42. Specifically, for example, monoclonal antibodies that recognize the amino terminal side of FPBβ1-42 and those that recognize the carboxyl terminal side may be prepared and used in the immunoassay.

In the method of the present invention, the monoclonal antibody used can be obtained by a method known per se (G., Köhler & C., Milstein, Nature, Vol. 256, p. 2495, 197
5). Hereinafter, the method for producing the monoclonal antibody used in the present invention will be explained in detail.

(A) Preparation of antigen-sensitized animal cells Animals are immunized with FPBβ,-42. Animals commonly used for immunization, such as mice, rabbits, and goats, are used as the immunized animals, and preferably those that have established myeloma cell lines as described below, such as Bal b/c mice. However, there are no particular limitations. There are no limitations on the immunization method either.

(B) Preparation of tumor cells Culture the required amount of tumor cells corresponding to the immunized animal used in Section A above. Tumor cells are e.g. 5P210/A g
14, which grow in a culture solution containing 8-azaguanine, but cannot grow in a culture solution containing hypoxanthine, aminopterin, or thymidine, i.e., as a cell line that can be selected depending on the culture solution components. Use established methods.

(c) Cell fusion Cell fusion is performed by known methods (G, Köhler & C.

Milstein, Nature, vol. 256.

495, 1975).

However, a method of electrical fusion may also be used, and is not particularly limited.

(D) Purification of anti-FPBβ, -42 antibody The monoclonal antibody produced by the hybridoma obtained by the above method is purified by a method commonly used for protein purification.

(E) Immobilization of anti-FPBβ1-42 antibody A known method can be used to immobilize the antibody used in the method of the present invention on a solid phase. Examples of the solid phase include polystyrene, polyethylene, polyvinyl chloride, and polycarbonate. , sepharose particles, latex, agarose, cellulose, polymethacrylate, etc. are used.

(F) Preparation of labeled antibody When anti-FPBβ1-42 antibody is labeled, the method of labeling and the method of detection thereof are not limited at all, and can be labeled and detected by known methods. When using something that can be detected indirectly as a label, such as an enzyme, examples of the labeling substance include peroxidase, β
Enzymes such as -D-galactosidase, alkaline phosphatase, urease, catalase, and β-glucuronidase are used. Things that can be directly detected as radioactivity, such as radioactive substances, include 3H, '25I, or 1
31■ et al., methods using fluorescent substances include, for example, fluorescamine, fluorella centiocyanate,
Tetrarhodamine isothiocyanate or the like is coupled to the monoclonal antibody by conventional methods. However, the labeling substance should not be limited to the above-mentioned substances.

Furthermore, when an unlabeled anti-FPBβ, -42 antibody is used, a labeled antibody that specifically recognizes the antibody or its immunoreaction product is used. In this case, the same labels as those described above are used.

Using the above-described immobilized anti-FPBβ1-42 antibody and labeled antibody, FPBβ1-42 in a sample can be measured by a sandwich assay. At this time, there is no particular limitation on the order in which the immobilized antibody, labeled antibody, and sample are brought into contact. Then, the generated immune reaction product may be separated from the free labeled antibody, and the label may be detected directly or indirectly.

(Effect of the invention) (1) FPBβ, -42 concentration in the sample is 0.1 to 20
It is possible to measure within the range of 0 ng/ml, and (2) compared to conventional methods, it is possible to measure a large number of specimens with extremely simple operations, in a short time, and with high sensitivity.

(Example) Detailed examples of the present invention will be described below. However, the present invention is not limited to these examples.

Example 1 Measurement of FPBβ1-42 by enzyme labeling method (1) Preparation of antigen-sensitized animal cells Ba1b/cv mice (female) were immunized with FPBβ1-42. For immunization, 100 μm of a sample prepared by emulsifying Freund's complete adjuvant and 100 μg/mouse of FPBβ1-42 was administered into the abdominal cavity of the mouse. Two weeks later, as a booster immunization, 100 μg of FPBβ-42/mouse emulsified with Freund's incomplete adjuvant was administered intraperitoneally to the mice. One week later, FPBβ1-42 was administered as the final immunization.
100 μg/mouse was added to phosphate buffered saline (0.85%
100 μm of the solution dissolved in 0.01% phosphate buffer containing NaCl, pH 7.2 (hereinafter referred to as PBS) was administered intraperitoneally. Three days later, the pancreas of the treated mouse was removed aseptically.

DMEMI○ml containing 15% calf fat serum (hereinafter abbreviated as 15% FCS) was aspirated with a syringe, and a 27-gauge needle was attached. The pancreas was placed in an ice-cold container and several injections were made with a syringe needle. A syringe needle was inserted and the mixture was refluxed to cause the pancreatic cells to flow out into the tissue. Filter the effluent through a nylon mesh, put it into a centrifuge tube, and incubate at 11,000 rpm.
The supernatant was discarded after centrifugation at p for 10 minutes. Red blood cells in a cell pellet were dissolved in a 0.15M ammonium chloride solution (1mM
Ethylenediaminetetraacetic acid-disodium salt (hereinafter referred to as EDT)
A) containing 0.01M carbonate buffer, pH 7,
2) Hemolyzed and centrifuged, and the cell pellet was further DM
The cells were centrifuged and washed twice with EM to obtain tile cells.

(n) Preparation of myeloma cells The myeloma cells were SP210-Ag14, an 8-azaguanine-resistant strain derived from Ba1b/C mice.
(hereinafter abbreviated as S P 210) was used.

20 μg/m] for one week before cell fusion
]] 8-Azaguanine was cultured in DMEM containing 15% F, and then DMEM containing 15% FC8 was used until cell fusion. Immediately before cell fusion, 5P210 was aseptically DM
Centrifugal washing was repeated twice for 10 minutes at 1100 Orp using EM.

(m) Cell fusion The pancreatic cells prepared in the above (1) and the myeloma cells prepared in the above (II) were mixed in a ratio of 5=1 and centrifuged (1000
rpm, 10 minutes) and the cell pellet was collected. The cell pellet was spread on the wall by tapping the centrifuge tube.

In it, 50% PEG (MERK) was heated to 37℃.
DMEM containing polyethylene glycol 4000)
0.5rnl of the solution was dropped little by little while rotating the centrifuge tube. After mixing by slowly rotating the centrifuge tube for 1 minute, DMEM that had been heated to 37° C. was added 10 times while rotating the centrifuge tube at a rate of 1 ml every 30 seconds. Next, slowly add 2ml of Fe2 and add 11000
rp and centrifuged for 10 minutes. Cell pellets with 15% FC5
and I X 10-4M Hyboxanthin, 4 X 10-
The cells were centrifugally washed twice (1000 rpm, 10 minutes) with DMEM (hereinafter abbreviated as HAT medium) containing 7M aminopterin and 1.6×10 −5 M thymidine. This culture solution was dispensed into 96-well plates (Fa 1 con #3042) in 200 μm portions at 5×10 5 cells/well. HAT medium was replaced every 3rd day with 100111/well changes. After 3 weeks, 1 x 10-'M hypoxanthine, 1°6 x 10-'M thymidine and 15% F.
DMEM containing C5 (hereinafter abbreviated as HT medium) was used for medium exchange.

(IV) Selection of hybridomas 100 cell colonies observed in 96-well plate
After the day, solid-phase enzyme immunoassay was performed, and the culture supernatant was treated with anti-F.
The presence of PBβ, -42 antibody was examined.

96-well immunoplate flat bottom (manufactured by Intermed)
50μ of FPBβ1-422 ug/m1
The mixture was dispensed into m/well and allowed to stand at 37°C for 1.5 hours. Remove the solution remaining in the well, and add a solution containing 0.04% Tween-20 in PBS (hereinafter referred to as PBS).
- After washing three times with T), 0.1% BSA was dissolved in P.
300 μl of BS-T solution was added to each well, and blocking treatment was performed at 37° C. for 1.5 hours. Next, 100 μm of the above culture supernatant was dispensed into each well and allowed to stand at 37° C. for 1.5 hours. After washing these wells three times with PBS-T solution, peroxidase-labeled rabbit anti-mouse IgG
Antibody (manufactured by Jackson Co., Ltd. >4000 times diluted was dispensed at 50 μl/well and left to stand at 37°C for 1.5 hours. PBS
-After washing three times with T solution, substrate solution (1,2% 2
,2-azinodi-(3-ethylbenzthiazoline sulfate)
- 0, 1M citrate buffer containing diammonium salts (ABTS) and 0.01% hydrogen peroxide (H2O2) (
100 μm of pH 5,1,)) was added to each well. 3
Leave at room temperature for 0 minutes, then add 200mM oxalic acid solution to 100%
μm was added to stop the enzyme reaction. The absorbance at 415 nm was measured, and anti-FPBβ was added to the wells where enzyme activity was observed.
, it can be seen that there are hybridomas that produce -4° antibodies. As described above, an antibody-producing hybridoma with a strong antibody titer was obtained.

(V) Preparation of conditioning medium In a syringe fitted with a 26 gauge needle, 10 ml of refrigerated 0.3
The 4M sucrose solution was blotted. A Ba1b/c mouse (male) was subjected to vertebrae dislocation, and the above solution was injected intraperitoneally in a sterile manner.

Within 5 minutes after injection, the intraperitoneal solution was collected using an ice-cold syringe with an 18-gauge needle attached to the left flank. Pour the recovered solution into an ice-cooled centrifuge tube, and proceed to step 11.
Centrifuged at 000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 15% FC8-DMEM was added to the cell pellet, stirred, and placed in a bush. 37℃, 5% carbon dioxide concentration, 95%
Cultured overnight in humidity. The culture supernatant was collected, filtered through a 0.22 μm membrane filter, and used as a conditioning medium.

(VI) Cloning Hybridomas capable of producing antibodies were made into a single clone using the limiting dilution method. 20 ml of HAT medium containing 1 ml of foundation medium prepared in section (V) above.
I have prepared 1. Adjust the hybridoma cells to be cloned into the above culture medium so that there is one cell in each well,
96-well plate with 200 μm/well (Falc
on#3042). For wells where cell colonies are observed from around the 10th day of culture, apply the above CIV
) Anti-FPBβ according to the solid-phase enzyme immunoassay method described in section
A hybridoma producing the 1-42 antibody was selected, and cloning was repeated again to establish a single hybridoma. Finally, 7 clones of Ibridoma were established.

(■) Purification of anti-FPBβl-42 antibody B a 1 b / c mice (female) 6 to 10 weeks old were intraperitoneally administered with 0.5 ml of bristane (2,6,10,14-tetramethylpentadecane). . Two weeks later, the anti-FPBβ1-42 antibody-producing hybridoma strain obtained in section (Vl) above was intraperitoneally administered to each clone at 2×1.
06 cells/mouse were transplanted.

Ascitic fluid produced around the 10th day was dropped into a centrifuge tube containing 1/20 volume of 0.2M EDTA by inserting an 18-gauge injection needle into the abdominal cavity. 400 centrifuge tubes
Centrifugation was performed for 10 minutes at Orpm, and the supernatant was collected. The collected supernatant was roughly purified according to a 50% ammonium sulfate precipitation fractionation method, dialyzed against a PBS solution containing 0.05% sodium azide, and purified by ion exchange chromatography and gel filtration. 5 under mercaptoethanol reduction
The purity of the antibody was confirmed by DS-polyacrylamide electrophoresis (12% T), which showed two bands, one heavy chain and one light chain.

By the above method, multiple types of monoclonal antibodies that specifically recognize FPBβ1-42 were obtained. The binding constants for each antibody ranged from 107 to IOIIM-1.

(■) Immobilization of anti-FPBβ1-42 antibody 3 μg/ml dissolved in 0.1 M sodium carbonate buffer (pH 9,6) in each well of an untreated microtiter plate (96-well Nunc plate, manufactured by Intermed) A solution of anti-FPBβ, -42 antibody (name A) derived from a mouse of
μm was added and incubated overnight at 4°C. Next, after removing the solution in each well and washing three times with PBS-T,
300 μm of PBS-T solution containing 0.1% BSA was added to each well, blocked at 4° C., and stored as it was.

(■) Preparation of horseradish peroxidase (HRP)-labeled antibody Add 1% 1-fluoro-
0.1 ml of ethanol solution of 2,4-dinitrobenzene
was added and reacted at room temperature for 1 hour. 0.0 in the solution
1.0rn3 of 6M sodium periodate was added and reacted for 30 minutes. 0.16 of unreacted sodium periodate
M ethylene glycol 1. After adding and removing Oml, it was dialyzed against 0°01M sodium carbonate buffer (pH 9,5). Next, 5 mg of mouse-derived anti-FPBβ, -42 monoclonal antibody (named B0 monoclonal antibody that recognizes a different antigen site from monoclonal antibody A) was added and reacted for 5 to 6 hours. 4 by adding 5 mg of sodium borohydride.
It was left overnight at ℃.

Thereafter, in order to remove unreacted sodium borohydride, the mixture was dialyzed against 10 mM sodium phosphate buffer (pH 7.1) containing 0.85% sodium chloride at 4° C. overnight with stirring. The above reaction product was added to TSK-Gel G-30.
It was purified by high performance liquid chromatography using 00SW (manufactured by Tosoh Corporation, trade name) to obtain an HRPIl-specific antibody.

(X) Quantification of FPBβ1-42 in the sample (
After returning the microtiter plate prepared by the method described in ① to room temperature and washing it with PBS-T solution,
20 standard samples containing Bβ, -42 were added to each well.
μm was added. Next, the HRP-labeled antibody obtained in this example (IX) was diluted with PBS-T solution, and 200 μm thick was added to each well.
were added one by one. After incubating at room temperature for 3 hours, the solution was removed and the plate was washed three times with PBS-T solution. Additionally, 0,1 containing 1.2% 2,2-azinodi-(3-ethylbenzthiazoline sulfate)-diammonium salt (ABTS) and 0.01% hydrogen peroxide (H2O2)
200 μm of a substrate solution consisting of M citrate buffer (pH 4,1) was added to each well and the enzyme reaction was allowed to occur for 30 minutes at room temperature, and then 100 μm of a 200 mM oxalic acid solution was added to stop the enzyme reaction. The above microtiter plate was heated at a wavelength of 415 nm and a control wavelength of 492 nm for each well.
Table 1 shows the results of measuring the absorption intensity of m using an automatic microtiter plate reader (manufactured by Tosoh Corporation, MPR-A4, trade name). As is clear from Table 1, it was confirmed that FPBβ1-42 in the sample could be quantified in the range of 0.1 to 200 ng/m+.

Table 1 patent obscene person Tosoh Corporation

Claims (1)

[Claims] A method for measuring fibrinopeptide Bβ_1_-_4_2 in a sample, comprising: (a) a monoclonal antibody immobilized on a solid phase that specifically recognizes fibrinopeptide Bβ_1_-_4_2; a sample;・Contact with a labeled or unlabeled monoclonal antibody that specifically recognizes fibrinopeptide Bβ_1_-_4_2 at a site different from that of the immobilized monoclonal antibody, and (b) prepare the unlabeled monoclonal antibody with (a). When an antibody is used, contact with the monoclonal antibody or a labeled antibody that specifically recognizes the immune reaction product generated in a, (c) remove free labeled antibody, and (d) label. 1. A method for measuring fibrinopeptide Bβ_1_-_4_2 in a sample, comprising the steps of directly or indirectly detecting and quantifying an immune reaction product.