JPS60233553A - Measuring method for fibrinogen and fibrin degradation products - Google Patents

Abstract

PURPOSE:To enable accurate measurement of the plasmin decomposed product of human fibrinogen or fibrin with a colorimeter by using an antibody prepd. by enzyme labeling of a specific antibody to the D or DD fraction in the plasmin decomposed product and said fraction deposited on an insoluble carrier. CONSTITUTION:The antibody, which reacts with the D fraction or DD fraction or the fraction holding the D fraction or DD fraction in the plasmin decomposed product of human fibrinogen or fibrin (hereafter expressed as FDP) but does not react with fibrinogen and is different in the antigenic determinants for recognizing such fractions with each other is obtd. by refining the crude antibody obtd. from the mouse ascites. The antibody prepd. by labeling such refined antibody by enzyme such as beta-D-galactosidase or glucose oxidase is prepd. The labeled antibody is added to the liquid mixture composed of specimen plasma, urine, etc. and is brought into reaction with the insoluble carrier adsorbed with the D or DD fraction and thereafter the quantity of the enzyme conjugated with the adsorbed carrier is measured. Said quantity is compared with a preliminarily obtd. calibration curve and the FDP is measured with good accuracy without the influence of the fibrinogen.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides fibrinogen using monoclonal antibodies;
This invention relates to a method for measuring fibrin degradation products.

In recent years, cases of death or worsening of underlying disease due to thrombosis/embolism have been increasing. For this reason, active fibrinolytic therapy is being performed. Blood coagulation and fibrinolytic systems exist as a defense mechanism for living trees, but abnormalities in these systems appear as disseminated intravascular coagulation syndrome (DIO). Fibrinogen and fibrin are usually used as clinical diagnostic methods. Conventionally, FDP has been measured using an agglutination reaction using sensitized latex using an anti-human fibrinogen antibody, but since this method uses anti-human fibrinogen antibody, serum or defibrinated specimens are always used as the specimen. had to use it.

However, polished specimens used to measure FDP often lack sufficient coagulation factors to be converted into serum, or fibrinogen itself is so low that sufficient coagulation does not occur. Coagulation itself may not occur in the blood of patients undergoing advanced anticoagulation therapy. Therefore, using serum as a specimen is extremely dangerous for clinical diagnosis. Although studies and research have been conducted to solve these problems, results sufficient for practical use have not yet been achieved.

The present inventors have completed the present invention as a result of intensive studies on a method for measuring FDP regardless of the amount of fibrinogen in plasma.

The present invention is an FDP measurement method using monoclonal antibodies obtained by the monoclonal antibody production method using hybridomas, which has been carried out in various fields in recent years.

The principle of the present invention is an enzyme immunoassay using enzyme-labeled antibodies. That is, FDP in the specimen and 08202.08 described in Japanese Patent Application No. 59-20842, which was filed earlier.
Both monoclonal antibodies of 204 were labeled with an enzyme (
After the antigen-antibody reaction with the labeled antibody (hereinafter referred to as labeled antibody), the remaining labeled antibody is transferred to a water-insoluble carrier coated with FDP.
After the second antigen-antibody reaction, the carrier is separated and the amount of enzyme bound to the carrier is measured, or a labeled antibody is added to the mixture of the analyte and the water-insoluble carrier, and the water-insoluble carrier is separated after a certain period of time. The amount of enzyme bound to the carrier can be measured using any of the following methods. Note that the amount of enzyme can be measured using a conventional method, and in any case, the amount of FDP in the subject can be calculated from a calibration curve prepared in advance.

In addition, β-D-galactosidase, peroxidase, alkaline phosphatase, glucose oxidase, etc. can be used as the enzyme to label the antibody, and the labeling method is enzyme immunoassay (Eiji Niban 9ishiyo, Igaku Shoin, 1
A labeled antibody can be obtained by a method known per se as described in (1982).

On the other hand, as a water-insoluble carrier, a piece of silicon, nylon, plastic, glass, a particle, a small sphere, a perforated flat plate, a test tube, etc. can be used, and methods for coating these carriers with the D fraction or the DD fraction include, for example. Of 1nic
a. Ohmica.

Acta, vol. 131, p. 101, 1988, same 1
Volume 85.

It may be prepared by a method known per se as described on page 261, 1983. Note that blood, plasma, serum, urine, etc. will be used as specimens that can be used in the present invention.

Reagent preparation method 1, purification of anti-FDP monoclonal antibody and F(ab'
) Crude antibodies were obtained from mouse ascites for labeling with the enzyme prepared in step 2. 50
The ascites was left at room temperature for 8 hours and then centrifuged to remove insoluble matter. The amount of IgG was 250 ■.

The centrifuged supernatant was salted out by adding the same amount of saturated ammonium sulfate.

The precipitate was dissolved in 10 mM Tris buffer at pH 8.0 and dialyzed against the same solution overnight. A column of DEAE-Sephacel (Pharmacia Sweden) buffered with the same solution (I
Apply the dialysis solution to the resin (100 ml resin volume per G zoolv), wash the column with the same solution, and then chlorinate it.
Linear concentration gradient from 0 mol/l to 0.2 mol/l (liquid volume used is 4 times the amount of column resin)
The protein was eluted by Mouse IgG was identified by the Ophthalony method using anti-mouse IgG antiserum, and the IgG fractions were pooled and salted out again. The mixture was filtered through a 1/X gel column using a Sephacryl S-AOO (Pharmacia Sweden) column with a diameter of 4.6 x 100 cm.

IgG was identified by the ophthalony method and further S and D S
- 90% pure Ig as determined by gel electrophoresis
I got G11200■. IgG is pooled and F(a b'
)2 was used as the raw material.

F(ab')2 was created by hydrolysis of pepsin.

That is, pepsin of 2 to 4% by weight was added to 2001vIgG adjusted to pH 4.2, and the mixture was left at 87° C. for 8 hours, and then the pH was adjusted to 8.0 to stop the digestion. 10
DEAE-Sephacel chromatography was performed by dialyzing against mM Tris pH 8.0 to obtain IgG. F(ab')2 is anti-mouse F(ab'),
and Ophthalony method using anti-Pc specific antiserum and 5D
8-Identified by gel electrophoresis. A trace amount of undigested IgG was removed by gel filtration. F(ab')2 fractions from gel filtration were collected and stored at 4°C. F(a
b'), it was 80 ■.

2. Labeling method using enzymes Enzyme immunoassay 2nd edition 1982 (Eiji Ishikawa et al.

According to Igaku Shoin), the details are as follows.

■ In the case of β-Kaisei Rautoshidase Fab', F(ab
') was set at 8 mp/mA', and 2 ml of it was dialyzed overnight against 0.1 M phosphate buffer pH 6-1 mM EDTA.
200μ of 2M mercaptoethylamine was added to the dialysate. Sephadex G25 was subjected to a reduction reaction at 87°C for 2 hours.
Gel filtrate with a column (φ14×80cR)
I got it. Concentrate to 2rnl and add 20μ4 of a DMF solution of 50my7/ul of N,N'-o-phenylene dimaleimide and react for ao'cao minutes.
ab'-maleimide. Gel filtration was performed using G25 to remove unreacted reagents, and the pH of the Fab'-maleimide solution was adjusted to 6.8. After adding 101Rg101Rβ-D-galactosidase (manufactured by Boehringer), adjust the liquid volume for 2 d and heat at 4°C.
The reaction was carried out for 40 hours. 10mM phosphate buffer pH 6,
5 (0.1M sodium chloride 1mM magnesium chloride-
Sephacryl 5 = 400 column (φ1.5X) equilibrated with 0.1% bovine serum albumin - 0.1% sodium nitride)
The β$7 lactosidase Fab' conjugate was separated from unreacted Fab' by 100 min).

■ For horseradish/peroxidase-Fab F
(ab')2 is 4.2 my/lnl, and its 0.
60μl of 0.4M mercaptoethylamine in 61nl
Add 8? A 13# ethanol solution of N-succinididyl-8-1 (2-pyridyldithio) propionate (Pharmacia) was added to 800 μl of lθ da/1.5 m/solution of peroxidase (manufactured by Toyobo Co., Ltd.) that had been subjected to a reduction reaction at ℃ for 90 minutes.
60μj of /at was added and reacted for a0 minutes at 25°C. F
Unreacted reagents were removed from both the reduced solution of (ab')2 and the pyridyl disulfide solution of peroxidase using a Sephadex G25 column to obtain F ab' and pyridyl dithioperoxidase, respectively.

2 my/0.2 ml of Fab', 1.8 of pyridyl dithioperoxidase, 10.2+1! A' was prepared and the two were mixed together and reacted at 28° C. for 20 hours while being dialyzed. Apply the dialysis fluid to a Sephacryl 5200 column (φ2 x 100 CIN) and place it on the Fab'-burner. -to 8, FD
Adsorption method of P onto water-insoluble carrier FDP-D, FDP-D
D was obtained by the method described in Japanese Patent Application No. 59-20842.

■ Adsorption onto polystyrene balls A polystyrene ball with a diameter of 61!11 (Shimiki Kagaku) was sonicated 8 times in deionized water to remove fine powder. FDP-
4μ of D! v! /ml 10mM Tris buffer p)(s,o-150mM sodium chloride-5rfi
The mixture was diluted with M calcium chloride (hereinafter referred to as Tris buffer) and gently mixed at a rate of 0.5 ml per ball at 4°C for 24 hours to allow adsorption. Unadsorbed proteins were thoroughly washed with Tris buffer and adsorbed with Tris buffer containing 1% bovine serum albumin-0 and 0.5% Tween 20. Unreacted bovine serum albumin-Tween 20 that had been stored at low temperature for 8 days was washed with Tris buffer and stored in the same solution.

■ Adsorption of FDP to polyvinyl chloride microborate
-DD in 10mM phosphate buffered saline (hereinafter referred to as PB)
The solution was diluted with S) to 0.5 μt/Ill.

Dispense 50μl of this into each well, leave it overnight at 4°C, remove the unreacted solution by suction, and incubate each well with z5oμ# 8 times.
% horse serum - 0,05%' [PB8 with 'ween20
Washed with. After washing, the water was shaken off and the surface was covered to prevent it from drying out and stored in a cold room.

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

■ The main conventional method is latex aggregation method.

Among these, the most common method is to examine the presence or absence of aggregation on a slide plate. The measured value generally lacks quantitative properties because it is expressed as the maximum dilution dust that indicates the flocculation ability. Further, in order to obtain the maximum diluted dust, dilution operations are usually required several times, which is complicated.

In the present invention, since a wide range of calibration curves can be prepared 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 value and is extremely dangerous for clinical diagnosis. The present invention provides anti-history l-'FDP-D/
Since it is a monoclonal antibody specific to DD and does not react with fibrinogen at all, not only serum but also plasma can be used, and under appropriate conditions, whole blood can also be used.

■ Attempts have been made to quantify latex aggregation by turbidimetry using special equipment, but this method is expensive and cannot be said to be widespread. Since the present invention is a colorimetric method, it can be quantitatively determined using a spectrophotometer and is a method that can be used very generally.

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

Example 1 Fab'-β-lactosidase measurement example using polystyrene balls (hereinafter referred to as balls).

200 μl of 100 μ4 Fab′-β′-H tosidase was added to the purified FDP-D using plasma as a test sample or a standard product in a test tube, and the mixture was reacted at 87° C. for zO minutes. Next, one ball was placed in the same test tube and the reaction was further carried out at 37°C for 20 minutes. The reaction solution was removed by suction using an aspirator, and the ball was washed twice with 2 ml of physiological saline. After wiping the water out of the bowl, add 500 μl of substrate solution (50 mM phosphate buffer solution, pH 7.2, containing lo mMO-nitrophenyl-β-D-galactoside and 100 mM β-mercaptoethanol) that has been kept at 37°C in advance. ) into a test tube containing 2rnl of 0.1
M sodium carbonate solution was added to stop the reaction. +o5iz
The absorbance of FDP-D was measured on the vertical axis, and the concentration of FDP-D on the horizontal axis was the standard curve value. Serum from the same patient was also measured in the same way.

(z-AlF bottle old λtoff Inventive method: FDP-D conversion value correlation between plasma and serum in the inventive method γ==o, 9os plasma,
It also correlates well with serum, indicating that it is not affected by fibrinogen even when plasma is used as a sample.

Example 2 Measurement example using Fab'-peroxidase and FDP-DD coated microplate.

Plasma or standard FDP-
After adding 5 ottll of DD, 50 μl of Fab'-peroxidase was added, and after reacting at 87° C. for 30 minutes, the reaction solution was removed by suction using an aspirator. Each well is 100μ
Washed twice with 1/1 saline solution. After washing, the substrate solution (p
21n in 1mJ of 50mM citrate buffer of H5,9
50 μl of aqueous solution containing orthophenylenediamine (No. 9) and hydrogen peroxide (0.84 μl) were added thereto, and the mixture was reacted at room temperature for 80 minutes. The reaction was stopped by adding 50 μl of 0.5M sulfuric acid. 60
Absorbance at 500 nm was measured using 0 nm t as a control. Example 1. Similarly, the vertical axis is absorbance, and the horizontal axis is FDP-D.
A standard curve was obtained using a semilogarithmic graph of D concentration. The measurement range was from 0.2 to 80 μjj−/ml. Example 1. The correlation with the measured value of the specimen was γ-09, which was good.

Claims (1)

[Scope of Claims] l) Reacts with human fibrinogen or a plasmin-decomposed fraction of fibrin, a DD fraction, a D fraction, or a fraction retaining a DD fraction, but not with fibrinogen. Antibodies that have enzyme-labeled antibodies with different antigenic determinants that recognize each other (hereinafter referred to as labeled antibodies) and D fractions or fibrinogen/fibrin degradation products characterized by DD (hereinafter referred to as FDP)
How to measure. 2) FDP in the specimen is subjected to an immunoreaction with the labeled antibody; 2) the remaining labeled antibody and the adsorption carrier are then combined; 1) the amount of enzyme in the labeled antibody-bound adsorption carrier is measured; claim 1) Measurement method described in section. 8) Claim 1) characterized in that the hindlimb adsorption carrier in which a labeled antibody is added to a mixed solution of the analyte and the adsorption carrier is separated, and the amount of enzyme bound to the adsorption carrier is measured. How to measure.