JPH0746104B2 - FDP measurement method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION “Industrial field of application” The present invention relates to a fraction that retains the D-dimer or the three-dimensional structure of D-dimer in a fibrin degradation product (hereinafter referred to as FDP) using one type of monoclonal antibody. Is related to the measurement method of. More specifically, a sample is obtained by immobilizing on a polystyrene latex a D-monomer or D-dimer in a plasmin degradation product of human fibrinogen or fibrin, or a fraction retaining the three-dimensional structure of the D-monomer or D-dimer It does not react with fibrinogen, X-fraction and Y-fraction, and does not aggregate even if it reacts with D-monomer or a fraction that retains the D-monomer steric structure, and retains the D-dimer or D-dimer steric structure. It is a quantitative method that is not affected by the amount of fibrinogen, the amount of X fraction, and the amount of Y fraction by using only the fractions that are used, and is a diagnostic method for the determination of the D-dimer in the sample and the fraction that retains the three-dimensional structure of the D-dimer. It is useful as a reagent.

“Prior Art” In recent years, the number of underlying diseases that cause death due to thrombosis / embolism has been increasing. Although the manipulation of thrombus formation is still largely unknown, clinical diagnostics for thrombus formation are evolving with increasing thrombosis. At present, an agglutination method using polystyrene latex particles sensitized with an anti-human fibrinogen polyclonal antibody is generally used for quantitative measurement of D-dimer in FDP. However, in this method, when fibrinogen, X fraction and Y fraction are mixed in the sample, the FDP becomes pseudo positive. In order to eliminate such a false positive reaction, it is required that the sample be completely defibrinogen defractionated and defractionated. However, such an operation is very complicated and is not suitable as an urgent clinical diagnostic agent. On the other hand, clinically, it has been expected to develop a method and a reagent for specifically and simply measuring D dimer or the like in FDP even in the presence of fibrinogen. The present inventors have conducted intensive studies to complete the present invention with the aim of overcoming these problems.

"Means for Solving Problems" That is, the present invention provides a method for measuring FDP using a monoclonal antibody, which comprises a D monomer in a plasmin degradation product of human fibrinogen and a D dimer or D monomer in a plasmin degradation product of human fibrin. Alternatively, one kind of anti-D-dimer / D-monomer monoclonal antibody having a property of reacting specifically with a fraction retaining the D-dimer three-dimensional structure but not reacting with fibrinogen, X-fraction, Y-fraction and _early D is immobilized. The modified polystyrene latex particles are brought into contact with a sample, the monoclonal antibody-immobilized particles and the fraction retaining the D-dimer or the three-dimensional structure of the D-dimer in the sample are selectively aggregated, and the aggregate is visually or spectroscopically analyzed. D-dimer or D in the sample
The present invention relates to a method for measuring FDP, which comprises semi-quantitatively or quantitatively detecting a fraction having a three-dimensional structure of dimer.

By the cell fusion method which has been applied in various fields in recent years, a novel anti-D dimer or D-monomer monoclonal antibody which does not react with fibrinogen, X-fraction and Y-fraction as described in "Production of Monoclonal Antibody" described later is obtained. By applying this to the latex agglutination method, it was possible to accurately measure the amount of D-dimer etc. in FDP in the sample even in the presence of fibrinogen, X fraction and Y fraction.

The principle of the present invention is that "one kind" of anti-D that reacts with the D-dimer and the fraction retaining the D-dimer steric structure or with the D-monomer and the fraction retaining the D-monomer steric structure.
This is a reverse passive agglutination reaction in which polystyrene latex particles sensitized with dimer and D-monomer monoclonal antibody are used to quantify the D-dimer in the sample and the fraction retaining the three-dimensional structure of D-dimer. That is, the polystyrene / latex particle suspension solution (pH 8.0) of the DD / D-1 monoclonal antibody described in "Production of Monoclonal Antibody" below.
Then, DD / D-1, anti-D dimer, D monomer monoclonal antibody-sensitized polystyrene latex particles are prepared.
The antibody-sensitized latex particles cause an antigen-antibody reaction with both the D-dimer and the fraction retaining the D-dimer steric structure or both the D-monomer and the fraction retaining the D-monomer steric structure. It has the unique property that it occurs only in the reaction with the fraction that retains the three-dimensional structure of the dimer. Therefore, after a certain amount of the sensitized latex and a certain amount of the sample were mixed on a slide plate or a microtiter plate for a certain period of time, the presence or absence of an agglutination image or its intensity was observed to retain the D-dimer and the three-dimensional structure of the D-dimer in the sample. The amount of fractionation to be performed can be determined. Alternatively, a fixed amount of the sensitized latex and a fixed amount of the sample are mixed, and the increase in absorbance after a certain period of time is spectroscopically measured at an appropriate wavelength to retain the D-dimer and the three-dimensional structure of the D-dimer in the sample. It is possible to quantify the amount of fractions to be applied. The polystyrene latex particles used in the present invention can be commercially available, and the particle size is 0.1 to 0.8.
It is preferable to use μm. In addition, plasma, serum, urine, etc. can be used as the test object that can be used in the present invention.

Fibrinogen, X fraction and Y used in the present invention
A novel anti-dimer, D-monomer monoclonal antibody that does not react with the fraction is obtained as follows.

Production of Monoclonal Antibody The anti-D-dimer D-monomer monoclonal antibody can be produced by culturing a novel mouse hybridoma in a medium or in the abdominal cavity of a mouse, respectively. The mouse hybridoma used here generally comprises spleen cells and mouse myeloma cells of a mouse immunized with D-dimer, and Khl
er and Milstein's basic method of cell fusion [nature 256
Volume 495 (1975)]. The details are as described in the following production example.

Further, as a medium for culturing the above hybridoma,
Any medium can be used as long as it is suitable for culturing hybridomas, and is preferably Dulbecco's modified Eagle's minimum essential medium (Dulbec
co's modified Eagle's minimum essential medium
Hereinafter referred to as DME. ) Fetal bovine serum, L-glutamine, L-
A medium containing pyruvic acid and an antidote (penicillin G and streptomycin) is used.

Cultivation of the above hybridomas is carried out at 5% CO ₂ concentration at 37 ° C. for about 3 days when carried out in a medium, and for about 14 days when cultured intraperitoneally in mice.

The above-mentioned monoclonal antibody can be separated and purified from the thus-produced culture medium or mouse ascites fluid by a method generally used for protein isolation and purification.

Examples of such methods include salting out with ammonium sulfate, ion exchange column chromatography using ion exchange cellulose, molecular sieve column chromatography using molecular sieve gel, affinity column chromatography using protein A-binding polysaccharide, dialysis, lyophilization and the like. is there.

Production Example 1 Method for preparing D-dimer and (DD) E The method for preparing D-dimer is mainly Stephanie A. Olexa and Andre.
i Z. Budzynski's method, (1978) Circulation, Suppl. 58 ,
119, Olexa et al., (1979) Biochim. Biophys. Ac.
It was performed according to ta 576, 39-50. Fibrinogen (mold, Sweden) 20 mg (10 mg / ml), human thrombin and calcium chloride, respectively, final concentration 10 units / ml,
Fibrinogen was converted to fibrin by adding 10 mM and reacting at 37 ° C. for 2 hours. Fibrin was separated from non-coagulable substances by centrifugation at 18,000 xg for 30 minutes. Fibrin is suspended in 20 ml of 0.15 M Tris-HCl buffer (pH 7.8) -5 mM calcium chloride-0.02% NaN ₃ solution. Human plasmin (1 unit / ml, Green Cross) was added to the suspension every hour for 0.
5 ml was added. After 10 hours, aprotinin (Mobay Chemica
(200 Corp.) was added to stop the decomposition reaction. capacity
Plasmin was removed by passage through a 10 ml Lysine Sepharose column.

Next, the flow-through is preliminarily 50 mM Tris-HCl buffer (pH 7.
5) The column (diameter 2.6 cm, length 90 cm) of Sepharose CL6B (Pharmacia, Sweden) equilibrated with -0.15 M sodium chloride-5 mM calcium chloride solution was packed. Molecular sieve chromatography was performed using the above solution. By the octelony method using molecular weight markers and anti-D, anti-E antiserum (Hoechst, Germany) (D
D) Fractions of E complex were identified and separated. The (DD) E complex thus obtained was treated with 3M urea-50 mM citric acid (pH 5.
5) Incubate at 37 ℃ for 4 hours in the solution. Next, the column was packed with Sepharose CL-6B (diameter 2.6 cm, length 90 cm) equilibrated with 50 mM Tris-hydrochloric acid buffer solution (PH7.4) -28 mM sodium citrate-0.1 M sodium chloride solution, and the above solution was added. Deployed in. D-dimer (DD) fraction and E, (E) by the octelony method using molecular weight markers and anti-D, anti-E antiserum
Fractions were identified and separated. A280nm = 2.0 D-dimer 10ml
Got The D-dimer thus prepared is used as an immunogen and as an antigen for an enzyme immunoassay (ELISA) for selecting anti-D-dimer monoclonal antibody-producing hybridomas.

Production Example 2 (a) Preparation of immunized spleen cells: The above D-dimer immunogen solution (A280nm = 2.0) was mixed with an equal amount of Freund's complete adjuvant until emulsified,
Immunization was performed by intraperitoneally administering 100 μl of the mixed solution (first immunization). After 30 days, the mice were intraperitoneally administered by the same method as described above (second
Immunity). 21 days after the second immunization, D-dimer immunogen solution (A280nm = 2.0) was diluted with an equal volume of physiological saline,
100 μ of the diluted solution was intravenously administered to the mouse (final immunization). Three days after the final immunization, spleen cells were taken out from the mouse and used for cell fusion.

(B) Cell fusion: The above-mentioned aseptically excised spleen is placed in a petri dish containing 5 ml of DME medium containing 10 to 15% fetal bovine serum. Next, the spleen is refluxed with about 15 ml of DME medium containing 10 to 15% fetal bovine serum to allow splenocytes to flow out, and this splenocyte suspension is passed through a nylon mesh. Collect the splenocytes in a 50 ml centrifuge tube and centrifuge at 500 xg for 10 minutes. The pellet thus obtained was mixed with 3-5 ml of hemolyzing solution (155 mM NHaCl, 10 mM KHCO
_3, 1mM Na ₂ EDTA pH7.0) was added and suspended. 5 at 0 ° C
The erythrocytes in suspension are destroyed after standing for ~ 10 minutes. Ten
Add ~ 20 ml of DME medium containing 10-15% fetal calf serum and centrifuge. The cell pellet obtained in this way
Wash with DME medium by centrifugation and measure the number of viable splenocytes.

On the other hand, 1 × 10 ⁸ of the above-mentioned spleen cells were added to approximately 2 × 10 ^{7 of} mouse myeloma cells (myeloma cells) SP2 / 0-Ag14 that had been cultured in advance, and mixed well in EME medium and centrifuged. Was performed (500 × g, 10 minutes). Aspirate the supernatant, loosen the pellet well, keep the temperature at 38 ℃, add 0.5 ml of 40% polyethylene glycol 4000 solution dropwise, and spin the centrifuge tube by hand for 1 minute to remove the polyethylene glycol solution. The cell pellets were mixed. Next, add 1 ml of DME medium kept at 38 ° C for 30 seconds and gently rotate the tube. After repeating this operation 10 times, 20-30 ml of DME containing 10-15% fetal bovine serum was used.
After adding the medium and centrifuging (500 xg, 10 minutes), removing the supernatant, the cell pellet was added to HAT medium containing 10 to 15% fetal bovine serum (DME medium with aminopterin 4 x 10).
^-7 M, thymidine 1.6 × 10 ^-5 M, hypoxanthine 1 × 10 ^-4 M)), washed twice by centrifugation, and then suspended in 40 ml of the above HAT medium. Add this cell suspension to 9
200 μm was dispensed into each well of a 6-well cell culture plate, and the culture was started at 37 ° C. in a carbon dioxide incubator containing 5% carbon dioxide. During the culturing, the culture medium in each well should be replaced with about 10
Hybridomas that grow in the HAT medium were selected by adding 100 μ of the above HAT medium, except 0 μ. HT medium containing 10-15% fetal bovine serum from day 8 (DME medium with thymidine 1.6 × 10 ^-5 M, hypoxanthine 1 × 10 5
^-4 M) was added, and the growth of hybridomas was observed.
Hybridomas producing anti-D monomer and D-dimer antibody were screened by the ISA method.

(C) Establishment of hybridoma The presence or absence of the produced antibody in the hybridoma culture supernatant was measured by the ELISA method. 96-well ELISA plate (Immulon I
I, Nippon Dynatech Co., Ltd.), 50 μm of the above-mentioned purified D-dimer solution (A280 nm = 0.05, diluted with physiological saline) was dispensed into each well and left at 25 ° C. for 2 hours. Next, after washing three times with 0.05% Tween 20-physiological saline, 50 μl of the culture supernatant was added to each well and reacted at 25 ° C. for 1 hour.

Next, 50 μl of peroxidase-conjugated anti-mouse antibody (Dako, Denmark) diluted 200 times with Tween 20-physiological saline was added to each well. After completion of the reaction, each well was washed 3 times with 0.05% Tween20-physiological saline, and 250 μl of a solution containing 0.5 mM aminoantipyrine, 10 mM phenol and 0.005% hydrogen peroxide was added to each well and reacted at 25 ° C. for 30 minutes. Then, the absorbance at 490 nm of each well was measured. As a result, antibody production was observed in 12 of the 192 wells.

For 96-well ELISA, which was sensitized with the above-mentioned antigen, to determine whether the anti-D-dimer antibody in the culture supernatant recognized by the above-mentioned ELISA method reacts with D-monomer, fragment X, fragment Y, fragment E and fibrinogen It measured using the plate by the same method as the above. As a result, one well of the culture supernatant in 12 wells reacted with D dimer and one well reacted with D monomer. The other 11-well culture supernatant reacted with all of D monomer, fragment X, fragment Y and fibrinogen.

The hybridoma was transferred to a 24-well plate of hybridoma in a well specifically reacting with D dimer and D monomer, and cultured in HT medium containing 10 to 15% fetal bovine serum for 4 to 5 days. Then, the presence or absence of production of anti-D dimer and D monomer antibody was confirmed again by the ELISA method, and then cloned by the limiting dilution method. In the limiting dilution method, a cell suspension prepared by diluting 5 hybridomas / ml in HT medium was pre-injected with 2 × 10 ⁴ peritoneal cells of normal BALB / C mouse per well.
100 μ was dispensed into each well of the 6-well plate. about
Ten days later, a hybridoma clone producing an anti-D dimer, D monomer antibody was screened by ELISA. As a result, 20 antibody-producing clones were obtained.
From these clones, clones with good proliferation, high antibody secretory ability, and stable clones were selected and recloned in the same manner as described above to prepare anti-D dimer, D monomer-specific antibody-producing hybridoma DD / D. -1 was established.

Production Example 3 (Production of Monoclonal Antibody) (In Vitro Method) Mouse hybridoma DD / D-1 was cultured in a DME medium containing 15% fetal bovine serum for 72 to 96 hours at 37 ° C. in a 5% carbon dioxide atmosphere. After centrifuging the culture (10 000 × g, 10 minutes), solid ammonium sulfate was gradually added to the supernatant to a final concentration of 50%. The mixture was stirred under ice-cooling for 30 minutes and then left for 60 minutes. After centrifugation (10000 xg, 10 minutes), the resulting precipitate was dissolved in a small amount of 10 mM phosphate buffer solution (pH8.0),
It was dialyzed against 0 volume of 10 mM phosphate buffer. This was loaded onto a column of DEAE-cellulose already equilibrated with 10 mM phosphate buffer. Elution of monoclonal antibodies
It was performed by a concentration gradient method between 10 mM phosphate buffer (pH 8.0) and 10 mM phosphate buffer (pH 8.0) containing 0.2 M NaCl.
Concentrate the eluted monoclonal antibody by ultrafiltration,
It was dialyzed against 0.1 M phosphate buffer (pH 8.0). The dialysate was passed through a column of goat anti-bovine serum 1 gG-Sepharose 4B to remove 1 gG bovine serum. Next, the flow-through solution was loaded onto a protein A-Sepharose 4B column equilibrated with 0.1 M phosphate buffer (pH 8.0). Purified anti-D dimer, D monomer specific resistance, D
D / D-1 was obtained.

(In vivo method) Pristane (2,6,10,14-tetramethylpentadecane)
0.5 ml was intraperitoneally administered to 10-12 week old BALB / C mice 1
The hybridoma DD / D-1 grown in vitro was inoculated into the abdominal cavity of mice on the 4th to 20th day at 2 × 10 ⁶ cells per mouse.

About 10 to 15 ml of ascites was obtained from one mouse. The antibody concentration was 5-10 mg / ml. Purification of the monoclonal antibody in ascites was performed by the same method as the above in vitro purification method (however, except the operation of passing a goat anti-bovine serum 1 gG-Sepharose 4B column).

Production Example 4 (Identification of immunoglobulin class and specificity of monoclonal antibody) Anti-D dimer, D monomer-specific monoclonal antibody DD / D-
Immune blobulin class 1 was performed by the Octelony immunodiffusion method. The results are as shown in Table 1.

Production Example 5 (Identification of Recognition Site of Monoclonal Antibody) The recognition sites of the monoclonal antibody, DD / D-1, were identified by Western blotting. Experimental operations are mainly performed by Bio-Rad, Inc., USA Zeta-Probe Blot
ting Membranes Instruction Manual.
The outline of the experimental operation is as follows.

Fibrinogen was treated with plasmin in the presence of Ca ²⁺ or EGTA, and fibrinogen degradation products after 30 minutes, 60 minutes, and 24 hours of reaction were subjected to SDS polyacrylamide electrophoresis in the presence and absence of DTT (dithiothreitol). Was done. Blotting and enzyme immunoassay were performed by the above-mentioned method, and the recognition site of the monoclonal antibody DD / D-1 was identified based on this result and the result of protein staining with SDS polyacrylamide gel by Coomassie Brilliant Blue G-250. Fibrinogen and purified D-dimer (DD) E were also treated in the same manner as above. The results are shown in Table 2.

"Examples" Hereinafter, the present invention will be further described with reference to Examples.

Experimental Example 1: Preparation of sensitized polystyrene latex particles A 10% suspension of polystyrene latex particles having an average particle size of 0.22 μm (manufactured by Japan Synthetic Rubber) was used in Tris buffer (20 mM Tris-HCl buffer-50 mM sodium chloride). After washing twice by a centrifugation method (30000 × g, 30 minutes), it was diluted with the same buffer solution to give a 1% suspension. After dialysis of the anti-human D-dimer, D-monomer monoclonal antibody DD / D-1 obtained by the above Production Example 3 against the above-mentioned Tris buffer,
The buffer solution was adjusted to a concentration of 0.6 mg / ml. Sensitization was performed by mixing a suspension of 1% polystyrene latex particles with the anti-human D-dimer, D-monomer monoclonal (DD / D-1) solution at a volume ratio of 1: 1 and then leaving the mixture at 25 ° C for 3 hours. Polystyrene latex particles were prepared. The unreacted monoclonal antibody that was not adsorbed on the polystyrene latex particles was removed by centrifugation (30000 xg, 30 minutes). The sensitized polystyrene latex is centrifuged (30000
Xg, 30 minutes) and then washed twice with Tris buffer as above, and then 0.1% serum albumin in 0.01% and 0.01% Tween 20
Was suspended in a Tris buffer containing 1% to give a 1% suspension. This one
% Suspension was used as the latex agglutination reagent.

Experimental Example 2: Measurement by slide agglutination method 20 μl of a latex agglutinating reagent and 40 μl of a test substance were mixed on a glass slide and shaken for 2 minutes to determine the presence or absence of agglutination. The results are shown in Table 3.

Experimental Example 3: Measurement by spectroscopic method Using LPIAL-1 (Mitsubishi Kasei Co., Ltd., Japan), the D-dimer or the three-dimensional structure of D-dimer in the plasma and serum samples from the normal person and the same person with the DIC patient is retained. The amount of the fraction was measured, and the correlation between the measured values of D-dimer and the like in the plasma sample and the serum sample was examined. Standard used DDE.

1% in a reaction container containing 50μ of sample (serum or plasma)
After injecting latex (50 μm) and Tris buffer (pH8.0) (200 μm), the absorbance change for 1 minute was measured at 700 nm, and DDE in the sample was quantified from the measured absorbance change value. The results are shown in Table 4 and FIG. The correlation coefficient r = 0.9944 showed a very good correlation. Note that in FIG. 1, N = 3
3, regression equation y = −33.5 + 0.9473x “Effect of invention” As is clear from the above, according to the present invention, even in a sample containing fibrinogen, X fraction and Y fraction, the aforementioned fibrinogen, X fraction, Y It has become possible to quantitatively and accurately and accurately measure the D-dimer in the sample FDP or the fraction retaining the three-dimensional structure of the D-dimer regardless of the fraction content.

[Brief description of drawings]

FIG. 1 is a graph showing the correlation between the concentration of D-dimer in plasma and the concentration of D-dimer in serum.

 ─────────────────────────────────────────────────── --Continued front page (56) References JP-A-59-183696 (JP, A) Chemical Abstracts, 99 (15), (10th Oct. 1983), p. 321-322 Thromb Haemastasis, 50 (2), (1983), P.P. 591-594 Thromb Res. , 31 (6), (1983), P. 767-778

Claims (3)

[Claims] 1. A method for measuring FDP using a monoclonal antibody, which comprises a D monomer in a plasmin degradation product of human fibrinogen and a D dimer or a D monomer and a D dimer steric structure in a plasmin degradation product of human fibrin. Contact with polystyrene latex particles immobilized with one type of anti-D-dimer / D-monomer monoclonal antibody, which has the property of reacting specifically with the antibody but not with fibrinogen, X-fraction, Y-fraction or _early D Then, the monoclonal antibody-immobilized particles and the D-dimer in the specimen or the fraction retaining the three-dimensional structure of the D-dimer are selectively aggregated, and the aggregate is visually or spectroscopically measured to obtain the D-dimer in the specimen. Or FDP characterized by semi-quantitatively or quantitatively detecting a fraction having a three-dimensional structure of D-dimer Measuring method. 2. The above-mentioned monoclonal antibody reacts with an antigen-antibody without aggregating with a D monomer having only one antigenic determinant or a fraction having a D-monomer steric structure, and a D-dimer having two antigenic determinants or The measurement method according to claim 1, which is a monoclonal antibody that reacts with an antigen-antibody reaction that selectively aggregates with a fraction having a D-dimer three-dimensional structure. 3. A method of spectroscopically measuring the formation rate of the aggregate, and quantitatively detecting the fraction having the D-dimer or the D-dimer three-dimensional structure in the sample. The measuring method according to the first or second range.