JPS60257364A - Measuring method of fibrinogen/fibrin degradation product - Google Patents

Abstract

PURPOSE:To measure a fibrinogen/fibrin degradation product (FDP) by using the monoclonal antibody obtd. by a method for producing a monoclonal antibody by hydriboma. CONSTITUTION:Either one of the monoclonal antibody is adsorbed on a water soluble carrier such as a slice or particle consisting of, for example, silicone (solid phase adsorbent antibody) and the other is labeled by enzyme such as peroxidase (enzyme labeled antibody). The specimen such as blood or plasma is brought into immunoreaction with the enzyme labeled antibody and the solid phase adsorbent antibody to form the composite material of the solid phase adsorbent antibody-antigen-enzyme labeled antibody. The enzyme activity in the composite material is measured and the quantity of the FDP in the specimen is measured from a preliminarily formed calibration curve.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring fibrinogen/fibrin degradation products using two types of monoclonal antibodies.

In recent years, cases of death or worsening of underlying diseases due to thrombosis/embolism have been increasing. For this reason, active fibrinolytic therapy is being performed. Blood coagulation and fibrinolytic systems exist as defense mechanisms of living bodies, and abnormalities in these systems appear as disseminated vasotropic coagulation syndrome (DEC). Usually, as a clinical diagnostic method, measurement of fifurinokene fibrin degradation products (hereinafter abbreviated as FDP) occupies an important position. Conventionally, I''DP has been measured by an agglutination reaction using a sensitized latex using an anti-human fifurinokene antibody, but this method uses either an antibody or an anti-human fifurinokene antibody.

It is necessary to always use serum or defibrinated specimens as specimens. However, patient samples used to measure FDP often lack sufficient coagulation factors to be converted into serum.

Fibrinogen itself may decrease and sufficient coagulation may not occur. In addition, coagulation itself may not occur in the blood of patients undergoing anticoagulant therapy. Therefore, using serum as a specimen is extremely dangerous for clinical diagnosis.

Various studies have been conducted to solve these problems.

We have not yet achieved results sufficient for practical use.

The present inventors investigated a method for measuring FDP regardless of the amount of fibrinokene in plasma and completed the present invention. The present invention is a method for quantifying FDP using monoclonal antibodies obtained by the monoclonal antibody production method using hybridomas, which has been practiced in various fields in recent years.

The present invention involves adsorbing one of the monoclonal antibodies 03202.03204 described in Japanese Patent Application No. 59-20842 to a water-insoluble carrier (hereinafter referred to as solid-phase adsorbed antibody), and labeling the other with an enzyme ( (hereinafter referred to as enzyme-labeled antibody). and.

The sample is immunoreacted with the enzyme-labeled antibody and the solid-phase adsorbed antibody to form a solid-phase adsorbed antibody-antigen-enzyme-labeled antibody complex, and the enzyme activity in the complex is measured. Calculate the amount of FDP in the sample from the calibration curve.

Note that a commonly used method can be used to measure enzyme activity.

As the enzyme used for the labeling of the present invention, β-D-galactosidase, peroxidase, alkaline phosphatase, glucose oxidase, etc. can be used, and the labeling method can be a method known per se (for example, enzyme immunoassay, (ed., Igaku Shoin, 1982). On the other hand, water-insoluble carriers include sections made of silicone, nylon, plastic, and glass.

Particles, small spheres, perforated flat plates, test tubes, etc. can be used, and adsorption methods include methods known per se (for example, J, Bi
ochem, Vol. 81, p. 1557, 1977). The sample used is blood.

Plasma, serum, urine, etc. can be used. .

Next, an example of the method for preparing the reagent of the present invention will be described.

Reagent Preparation Method (1): Purification of anti-FDP monoclonal antibody Crude antibody was obtained from mouse ascites.

5 oml of ascites was left at room temperature for 3 hours, and then centrifuged to remove insoluble matter. The amount of IgG was 250η. The centrifuged supernatant was salted out by adding the same amount of saturated ammonium sulfate. The precipitate is p
It was dissolved in 10mM H8,0)'JS buffer and dialyzed against the same solution overnight. A column (IgG
After applying the dialysis solution to 200 mg of resin (100 ml of resin) and washing the column with the same solution, the θ of the sodium chloride column was
Proteins were eluted using a linear concentration gradient from mol/l to 0.2 mol/l (4 times the amount of column resin was used in each case). Mouse Tg() was identified by the Ophthalony method using anti-mouse IgG antiserum, and the IgG fractions were pooled and salted out again. yJ 4.6 x 100cm
Gel filtration was performed using a column of Sephacryl S-3OO (manufactured by Pharmacia, Sweden). IgG was identified by the off-clone method, and the purity was verified by 5DS-gel electrophoresis, resulting in a purity of 90% (71) IgG.
200 mg was obtained.

2. Method for adsorption of antibodies onto water-insoluble carriers ■ Adsorption onto polyvinyl chloride microplates 032.02 described above was used as the clone.

08202 IgG was added to 5omM phosphate buffer (pH 72).
)-Physiological saline (PBS) was used to adjust the concentration to lag-/ml, and 50 μβ of the solution was dispensed into wells of a microplate. After standing at a low temperature for 24 hours, PBS containing 1% bovine serum albumin-005% Tween20 (BSA-P
BS) and washed 8 times with 100 μβ each.

■ Adsorption to polystyrene particles Clone 03204-, previously described, was used.

03204 IgG was diluted with 50mM carbonate buffered (pH 9,6)-physiological saline to a concentration of 4mO,D, 8o/ml. Polystyrene particles with a diameter of 67'rrI'n (manufactured by Tanezu Kagaku Co., Ltd.) that have been cleaned in advance by ultrasonic treatment
5 rnl of diluted antibody was added to each plate and mixed well at low temperature for 24 hours. Unadsorbed protein was removed by suction using an aspirator, and the plate was washed twice with physiological saline.

Thereafter, the unreacted hole surfaces were coated with BSA-PBS. The mixture was left at a low temperature for 8 days, and the BSA was removed with PBS, and the mixture was stored in PBS.

8 Enzyme labeling method for antibodies■ In the case of β-D-caractosidase 0812 0820
2 F(ab'), 3 In97m1! Toshi, Part 2
ml of 0.1M phosphate buffer (pI-16) -1mM
Dialysis was carried out against EDTA overnight, and 200 μl of 02M mercaptoethylamine was added to the dialysate. Reduction reaction was carried out at 37°C for 2 hours, and Sephatex G-25 column (961,2
Fab' was obtained by gel filtration using 3Q cm).

Concentrate to 2 ml and 50 m9/rnl of N, N'-o-
DMF solution of phenylene dimaleimide zom7! Add.

The mixture was reacted at 30°C for 80 minutes to obtain Fab'-maleimi 1-. The pH of the Fab'-maleimide solution was adjusted to 63 by gel filtration using Sephadex G-25 to remove unreacted reagents. 1
After adding 0 mg of β-D-galactosidase (manufactured by Behrin Riki Co., Ltd.), the liquid volume was adjusted to 2 m 7 and reacted at 4° C. for 40 hours. 10mMIJ acid buffer 1) H6,5(0,
1M sodium chloride, 1mM magnesium chloride, 0.1
% bovine serum albumin.

Sephacryl S-40 equilibrated with 01% sodium nitride)
β-
D-galactosidase Fab' conjugate was removed from unreacted Fal
)' separated from.

■Horserradish peroxidase-o82o4Fab'
In the case of 03204, F(ab') is 4.2 my/ml! Bind and add 0.4M mercaptoethylamine 60 to 06ml.
10 mg 71.5 ml solution of peroxidase (manufactured by Toyobo Co., Ltd.) and ethanol of N-succinimidyl-8-(2-pyridyldithio) propionate (manufactured by Pharmacia). 60 μl of solution 13~/ml was added and reacted at 25° C. for 80 minutes. Unreacted reagents were removed from the reduced solution of F(ab')2 and the pyridyl disulfide solution of peroxidase using a Sephadex G-25 column to obtain Fab' and pyridyl dithioperoxidase, respectively. Prepare Fab' to 2×70.2 ml and pyridyl dithioperoxidase to 1.8 μ/0.2 rnl.
Both were mixed and reacted at 28°C for 20 hours while being dialyzed. The dialyzed fluid was applied to a Sephacryl S-'200 column (Da 2x100) to purify the Fab'-peroxidase conjugate.

4. Preparation of standard FDP was carried out by the method described in the patent application (Japanese Patent Application No. 59-20842).

Next, the advantages of the present invention will be listed in comparison with the conventional method.

■Since a wide range of calibration curves can be created in advance, quantitative performance is excellent and sample dilution is not required.

■All conventional antibodies show cross-reactivity with human fibrinogen. Therefore, if even a small amount of fibrinogen remains or is mixed in the sample, it will greatly affect the measured values and is extremely dangerous for clinical diagnosis.

The present invention is a monoclonal antibody specific to anti-human FDP D/DD, and since it is an antibody that does not react with fibrinogen at all, it can be used with serum as well.

Plasma and whole blood can also be used.

The present invention will be explained below with reference to Examples.

Example 1 A 03202 adsorption microplate and a peroxidase label 03204 are used.

Measurement method: 50 μl of standard FDPD or plasma was added to a well of a microplate, and then 50 μl of labeled antibody was added. After reacting for 2 hours at room temperature, the wells were washed 8 times with 100 μl each of BSA-PBS.

50μd of substrate solution (2mg of orthophenylenediamine in 1ml of 50mM citrate buffer pH 5.9 and 08
4μ of hydrogen peroxide solution) was added thereto, and the mixture was allowed to react at room temperature for 80 minutes. The reaction was stopped by adding 50 μl of 0.05M sulfuric acid, and the absorbance was measured at 500 nm with 600 nm as a control.

As a result, it was possible to measure in the range from ■ to 200 μy-7 ml. In addition, serum from the same patient was similarly measured instead of plasma.

result Standard curve value Sample measurement value (FDP-D conversion value) correlation Plasma-serum; γ=0.9 [B As shown above, the plasma and serum values were in good agreement.

Kokichi was shown to be quantitative and to specifically measure FDP without being affected by fibrinogen.

Example 2 Measurement method using L1204 polystyrene particles and β-D-galactosidase labeled 03.202 50 μl of a standard or plasma and 500 μl of labeled antibody were added to a test tube, and one polystyrene particle with a diameter of 6 mm was added to the tube. 37
After the antigen-antibody reaction was carried out at ℃ for 30 minutes, the reaction solution was removed by suction using an aspirator. After washing the particles twice with 2 m/+ physiological saline, a 500 μ
1 of substrate solution (50mM, pH 7.2, containing 10mM MO-nitrophenyl-β-D-galactoside and 100mM β-mercaptoethanol.

The mixture was transferred to a test tube containing phosphate buffer) and an enzyme reaction was performed at 37°C for 15 minutes.

As a result, it was possible to measure up to 1 to 200 μg/ml, and the measured values were in good agreement with Example 1.

Claims (1)

[Claims] ■) Reacts with human fibrinogen or the plasmin-digested product of fibrin, the DD fraction, or the D fraction or both fractions that retain the DD fraction, but has the property of not reacting with fifrinogen, and with each other. Fibrinogen/fibrin degradation characterized by adsorbing one of two types of monoclonal antibodies that recognize different antigenic determinants onto a water-insoluble carrier, and immunoreacting each antibody labeled with an enzyme with the analyte. Product measurement method.