JPH0461639B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel method for producing 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 the living body,
These abnormalities are called disseminated intravascular coagulation (DIC)
It appears. Fibrinogen and fibrin degradation products (FDP) are usually used as a clinical diagnostic method.
The measurement of occupies an important position. Conventionally, FDP is generally 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 dehydrated blood is always used as the specimen. It is necessary to use fibrinated specimens. However, patient specimens used to measure FDP often lack sufficient coagulation factors to make into serum, or fibrinogen itself is so low that sufficient coagulation does not occur. Furthermore, coagulation itself may not occur in the blood of patients undergoing anticoagulant therapy. Therefore, using serum as a specimen is extremely dangerous in terms of clinical diagnosis. Although research has 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 extensive research into a method for measuring FDP regardless of the amount of fibrinogen in plasma. In the present invention, mice are immunized with D fraction or DD fraction obtained by purifying human fibrin or purified human fibrinogen degraded with plasmin by gel filtration and ion exchange chromatography using as an antigen. Mouse antibody-producing cells and mouse myeloma cells are fused, and the culture solution of the resulting fused cells is reacted with purified D fraction or purified DD fraction, and then fibrinogen is reacted with the D fraction or DD fraction in a solution. D fraction, DD fraction, This is a method for producing a monoclonal antibody that reacts with fractions and Y fraction, but does not react with human fibrinogen and E fraction. Each step of the present invention will be explained. Human fibrinogen is purified by ion exchange chromatography and gel permeation chromatography. After converting this purified fibrinogen into stabilized fibrin using human thrombin and factor factor, the non-coagulable substances and fibrin are separated by centrifugation, the fibrin is then degraded with human plasmin, and subjected to gel filtration and ion exchange chromatography. After obtaining FDP DD by graphie or directly digesting purified fibrinogen with plasmin,
By gel filtration and ion exchange chromatography
Obtain FDP D. Mice are immunized with FDP DD or FDP D thus obtained, and after a certain period of time, the spleens of the mice are removed. The excised spleen cells are fused with myeloma cells in the presence of polyethylene glycol to form hybridomas of cells producing specific monoclonal antibodies against FDP D and DD, or fibrinogen or fibrin decomposition products containing D. The selection of antibody-producing hybridomas is based on
Usually, this is carried out by ELISA in which hybridoma supernatant is reacted with immobilized antigen, but in the present invention, it is carried out by reacting hybridoma supernatant with purified D fraction or purified DD fraction and then with fibrinogen in a solution. . When a solid-phase antigen is used to select hybridomas, it is impossible to select hybridomas that produce antibodies whose antigen recognition site is in the binding site with the solid phase, but the present invention does not have this drawback. . The antibody may be obtained by culturing this hybridoma in a medium, or by injecting it into the peritoneal cavity of a mouse and producing the antibody in ascites fluid (in vivo culture). The antibody thus produced is purified by ammonium sulfate salting out, ion exchange chromatography, or gel filtration to obtain a highly pure antibody. This antibody was treated with cyanodiene bromide-activated Sepharose (Pharmacia).
Various purified proteins were immobilized and packed in a column and allowed to flow down. The adsorbed proteins were eluted with 4M urea-containing 50mM glycine buffer pH 2.8 and analyzed by electrophoresis based on the difference in molecular weight, resulting in FDPD, DD, X, and Y. The antibody clearly reacted with fibrinogen and FDPE, and was found to be a monoclonal antibody specific to the D fraction and the fraction containing the D fraction. In addition, normal human plasma was allowed to flow down and the same procedure was performed, but no reacting substances were found. A protein that can be considered as FDP was detected. Because of the above properties, by using the monoclonal antibody obtained in the present invention, FDP can be measured regardless of the amount of fibrinogen in the sample. Therefore, specimens include blood, synovial fluid,
This is an excellent method as urine etc. can be used. Examples will be described in detail below, but the present invention is not limited thereto. Example 1 (1) Method for preparing monoclonal antibodies (a) Purification of fibrinogen Human fibrinogen was purchased from Kabi (Sweden) and purified by the following method. 1 g of fibrinogen was dissolved in 100 ml of 50mM Tris buffer (PH8)-5mM sodium citrate solution and dialyzed against the same solution overnight. The dialysis fluid was centrifuged at 10,000 revolutions/min for 10 minutes to remove insoluble matter, and the supernatant was equilibrated with the same solution.
Apply to a column (diameter 3 cm, length 25 cm) of Cefacel (Pharmacia, Sweden), and then add sodium chloride to the column (diameter 3 cm, length 25 cm).
Proteins were eluted using a linear concentration gradient method up to 0.2M. It was analyzed by SDS/electrophoresis, and a fibrinogen fraction containing very few other impurity proteins was collected, and polyethylene glycol (PEG) with a molecular weight of 4,000 was added to the final concentration of 4.5% to precipitate the fibrinogen. The precipitate was centrifuged (10,000 revolutions/min,
10 min) and 50mM Tris buffer (PH7.2)
-Dissolved in 50mM sodium chloride-5mM sodium citrate solution to make 70ml. This fibrinogen solution was dispensed into plastic test tubes in 70 ml portions and stored at -80°C. (b) Preparation of FDPDD Fibrinogen 70mg (10mg/
ml) at 37°C, human thrombin, human factor 13 (both from Midori Jujisha) and calcium chloride were added to final concentrations of 10 units/ml, 1 unit/ml, and 10 mM, respectively, and incubated at 37°C for 3 hours. The fibrinogen was converted to cross-linked fibrin (hereinafter referred to as fibrin) by incubation. Fibrin and non-coagulable substances were separated by centrifugation at 13,000 rpm for 20 minutes. Fibrin in 20ml of 50mM Tris buffer (PH7.2)
Suspend in 150mM sodium chloride-10mM calcium chloride solution and add 0.25 units/ml human plasmin (Midori Juji) once every hour at 37°C.
Fibrin was degraded by adding 5 ml each. Insoluble fibrin became almost undetectable after about 8 hours, so trasylol (Bayer, West Germany) was added to a final concentration of 100 units/ml to stop the decomposition reaction. Plasmin was removed by passing through a 5 ml lysine-Sepharose column. The flow-through was stored at -80°C overnight. The lysate was thawed and applied to a Sephacryl S-300 (Facimacia, Sweden) column (diameter 2.6 cm, length 100 cm) equilibrated with a 50 mM Tris buffer (PH8) - 500 mM sodium chloride - 10 mM calcium chloride solution. Gel filtration developed with equilibration solution was performed. The fractions were fractionated and the high molecular weight FDP, DD, D, and E fractions were analyzed by SDS electrophoresis and the Ouchterlony method using anti-D, anti-E, and antiserum (all from Hoechst, West Germany).
Since the DD fraction was eluted together with a portion of the E fraction, it was collected as the DD and E fractions and dialyzed overnight against a 50 mM Tris buffer (PH8)-5 mM calcium chloride solution. DEAE- equilibrated with the same solution
Sefacel column (diameter 2.5cm, length 20cm)
The dialysis fluid was applied to the tube, and the DD fraction was separated into a non-adsorbed fraction, and the E was separated into an adsorbed fraction.
The purified DD fraction was diluted with 10mM Tris buffer (PH
7.2) - Dialyzed against 150mM sodium chloride-5mM calcium chloride solution, dialysate is 500μg
The mixture was transferred into small test tubes and stored at -80°C. (2) Immunization Mix 20 μg of DD fraction with 0.2 ml of complete Freund's adjuvant and inoculate 7-week-old Balb/
injected into the peritoneal cavity of C mice. Two weeks later, 20 μg of the DD fraction was thoroughly mixed with 0.2 ml of incomplete Freund's adjuvant (hereinafter referred to as IFA) and injected intraperitoneally. DD fraction 20 μg/IFA 0.2 ml every 2 weeks
was immunized by intraperitoneal injection. (3) Cell fusion Two weeks after the final immunization, inject 50 μg of DD fraction into
Mix with 0.1ml of physiological saline and inject into the tail vein 3.
After a few days, the spleen is removed and loosened, and then thoroughly washed with culture medium (RPMI1640). Mix 10 ⁸ of these washed splenocytes with 10 ⁷ mouse sp2/0 Ag14 myeloma cells, which were also thoroughly washed with medium, and add 0.5 ml of 50w/v% PEG1540 to the medium.
was gradually added dropwise and mixed for 1 minute. 8 ml of medium was gradually added to dilute the PEG and stop the reaction.
Cells were collected under centrifugation conditions of 1500 rpm for 5 minutes and washed once with 2 ml of medium. Cells were suspended in 30 ml of 10% FCS supplemented medium, 0.1 ml was dispensed into each well of a 96-well microplate, and incubated at 37° C. in the presence of 8% CO ₂ . HAT medium (10 ^-4 M hypoxanthine, 4x
A 10% FCS supplemented medium containing 10 ^-7 M aminopterin and 1.6 x 10 ^-5 M thymidine) was dispensed in 0.1 ml portions every day for 3 days from the day after the fusion. After further incubation for 3 days, 0.2 ml of the culture supernatant from each well was removed and replaced with 0.2 ml of HT medium (HAT medium with aminopterin removed). This operation was repeated three times. (4) Selection and cloning of fused cells (a) Selection Anti-D antiserum was diluted 5,000 times with phosphate buffer (PH7.2)-physiological saline (hereinafter referred to as PBS) and aliquoted in 50μ aliquots into a 96-well microplate. Pour at 4℃
was fixed overnight. PBS with 1% bovine serum albumin (BSA) and 0.05% Tween20
Wash the wells thoroughly with BSAPBS (hereinafter referred to as BSAPBS), add fibrinogen adjusted to 10 μg/ml with BSAPBS to one plate, and add the same amount of fibrinogen to the other plate.
50μ each of DD adjusted to 2.5μg/ml
The solution was aliquoted and allowed to undergo antigen-antibody reaction overnight. Plates were washed extensively with BSAPBS. 50μ of the culture supernatant was added to the fibrinogen-reacted plate and allowed to react at 37°C for 1 hour. Furthermore, add 50μ of fibrinogen 0.2mg/ml solution37
The reaction was maintained at ℃ for 1 hour. Add 50μ of this reaction mixture to
The DD fraction was reacted and added to the washed plate, and the remainder was discarded. The DD fraction was added to the reacted plate and reacted at 37°C for 1 hour. Each of the two types of plates
After thorough washing with BSAPBS, 50μ of an anti-mouse IgG goat antibody-peroxidase conjugate diluted 1000 times was added. After reaction at 38℃ for 1 hour
BSAPBS was washed with water, and an enzymatic reaction was performed using hydrogen peroxide and orthophenylene diacin as substrates. Activity was expressed as absorbance at 500 nm. Measurement examples are shown in the microplate table below.

[Table] The upper row shows the amount before absorption with fibrinogen.
The absorbance at 500 nm is shown, and the lower row shows the absorbance after absorption. Normally, the absorbance of each well decreased after absorption, as in A12 and F7. In other words, it was a hybridoma that produced antibodies that also reacted with fibrinogen. Even after absorption, such as A5, C4, and E3, although there are few cases,
There were also hybridomas that produced antibodies that strongly reacted with the DD fraction. Since there were five wells containing such cells, we decided to clone them. (b) Cloning The thymus gland of a 4-week-old young Balb/c mouse was taken out and thoroughly suspended in medium. Collect cells by centrifugation and add 5 x ¹⁰⁶ cells to 10% FCS supplemented medium.
The cells were resuspended to a concentration of cells/ml. The mixture was dispensed into a 96-well microplate at 0.2 ml/well and used as a feeder layer. 10 cultured cells of 5 types
%FCS supplemented medium to 10 cells/ml, and dispensed 01 mm into each well. Since the number of cells increased sufficiently after 10 days of culture, the same dilution-dispensing-propagation process was performed four times. The culture supernatant was analyzed for anti-DD, anti-D, and anti-fibrinogen activities at each cloning, and cells that had undergone the fourth cloning were determined to be monoclonal antibody-producing cells. Example 2 Preparation of FDP.D 70 mg (10 mg/
ml) at 37°C, and the final concentration of calcium chloride was 10.
The fibrinogen was added to give a concentration of mM, and while kept at 37°C, 1 ml was added every hour for 5 times to decompose fibrinogen for 8 hours. Trasylol was added to a final concentration of 100 units/ml to stop the reaction, and the mixture was passed through a 5 ml lysine-Sepharose column. The passed-through solution was stored at -80°C overnight, thawed, and transferred to Cephacryl S-300 used in (b).
It was applied to a column and passed through a gel. The fraction was analyzed by SDS electrophoresis and Ouchterlony's method, and the D fraction was identified. Fraction D was eluted partially overlapping with fraction E, so it was collected and dialyzed against 50mM Tris buffer (PH8)-5mM calcium chloride solution. Apply it to a DEAE-Sephacel column (diameter 2.6 cm, length 20 cm) equilibrated with the same solution, and
The fraction was separated into a non-adsorption part, and the E fraction was separated into an adsorption part.
Purified D fraction is 10mM Tris buffer (PH7.2)
Dialysis was carried out against -150mM sodium chloride-5mM calcium chloride solution, and the dialysate was dispensed into small test tubes in 500μg portions and stored at -80°C. D obtained in this example instead of the DD fraction in Example 1
Using the fractions, operations (2) to (4) were performed in exactly the same manner to obtain monoclonal antibody-producing cells. Example 3 The immunological classification of the five fibridoma strains established in Example 1 is shown below.

[Table] In order to determine the differences in the antigenic determinants recognized, each antibody was labeled with peroxidase and the DD fraction was subjected to a competitive reaction with a free antibody, and the recognition site was determined based on the presence or absence of inhibition. . The results are shown next.

[Table] +〇 Inhibition -〇 No inhibition From the above results, 03202, 03203 (03204, 03205,
03206) has been grouped. Although the antigen recognition site of 03203 is different from the other two groups, the enzyme-labeled product seems to have weaker reactivity or to inhibit binding to antigens that have already bound to other antibodies. The types of FDP recognized by the three groups are shown below.

[Table] + React - Not react None of the three groups reacted with fibrinogen and E, but reacted with other D and FDP containing D.
It also reacted with patient samples that tested positive using a commercially available FDP measurement kit. Example 4 The immunological classification of the two hybridoma strains established in Example 2 is G class 2a and 1, and L
Both chains were K. Investigations were conducted in the same manner as in Example 3, and it was found that both recognized different antigenic determinants. Also, Example 2
We investigated the differences between this monoclonal antibody and
It turned out that they correspond to 03202 and 03204, respectively.

Claims (1)

[Scope of Claims] 1. A mouse is immunized with the D or DD fraction obtained by purifying human fibrin or a plasmin-degraded product of purified human fibrinogen by gel filtration and ion exchange chromatography using as an antigen, Antibody-producing cells from immunized mice and mouse myeloma cells are fused, and the culture solution of the resulting fused cells is reacted with purified D fraction or purified DD fraction, and then with fibrinogen in a solution to form D fraction or DD. It is characterized by selecting a fused cell that reacts with the fraction but not reacting with fibrinogen, and then cloning to obtain a monoclonal antibody-producing cell, and further culturing the antibody-producing cell.
A method for producing a monoclonal antibody that reacts with D fraction, DD fraction, X fraction, and Y fraction, but does not react with human fibrinogen and E fraction. 2. The method for producing a monoclonal antibody according to claim 1, wherein the monoclonal antibody obtained belongs to the following immunological classification. (1) Immunoglobulin class IgG (2) Subclass 2a, 2b or 1 (3) Light chain κ.