JPH0940700A - Monoclonal antibody - Google Patents

Abstract

(57) [Summary] [Object] To provide a specific monoclonal antibody against a D-dimer in a rat thrombotic degradation product and an enzyme immunoassay using the antibody. [Structure] A molecular weight of 74 kg, which is produced by fused cells of mammalian cells and myeloma cells immunized with the γ-γ chain separated from rat D-dimer and strongly recognizes rat D-dimer than rat fibrinogen. Dalton's monoclonal antibody and enzyme immunoassay using it as an antibody. [Effect] Since the monoclonal antibody of the present invention can recognize rat D-dimer more strongly than rat fibrinogen, it is possible to measure D-dimer in rat blood by the enzyme immunoassay using this, and the rat is used as an experimental animal. It is very useful for the development and research of thrombolytic agents.
Moreover, this monoclonal antibody can be mass-produced by culturing the fused cells.

Description

Detailed Description of the Invention

[0001]

The present invention relates to rat D-dimer (D
-Dimer), a monoclonal antibody, a method for producing the same, and a method for using the same.

[0002]

2. Description of the Related Art Since ancient times, many diseases caused by thrombosis, such as pulmonary embolism and myocardial infarction, are life-threatening and have always been at the top of the causes of death. In the formation of a thrombus, the blood coagulation system in the living body is activated, and a fibrin thrombus is formed by thrombin generated as a result of the blood coagulation mechanism. This fibrin thrombus triggers the activation of fibrinolysis, and fibrin is hydrolyzed by plasmin to release soluble fibrin degradation products into the blood. These degradation products are collectively referred to as FDP (fibrin / fibrinogendegr
It is called "adduction products". In recent years, a method for measuring several markers showing the activity of plasma coagulation and fibrinolytic system has been clinically utilized, and among them, D-dimer which is a specific product when fibrin is decomposed by the action of plasmin is used. Measuring blood coagulation
It is important as one of the parameters of the state of accelerated fibrinolysis, and an enzyme immunoassay method using a human D-dimer assay reagent is currently in practical use.

[0003]

In developing a novel thrombolytic agent, it is not permitted to immediately administer the drug candidate substance to a patient because of its effects.
First of all, it is indispensable to create an animal pathological model that is close to the human pathological condition and study the drug efficacy using the model animal. But,
Since the anti-fibrinogen / fibrin antibodies that have been reported up to now are produced using human-derived antigens, thrombolytic products in model rat plasma cannot be accurately measured. Therefore, it uses a monoclonal antibody that reacts specifically with a thrombus-degraded product, does not require special facilities or instruments like the method using a radiolabel, and is extremely simple and safe with no concern about half-life or waste. There is a need for a method for detecting thrombotic degradation products in model animals.

Conventionally, rat FDP was purified by gel filtration using Sephadex G-100 and by isoelectric focusing, and it was shown that it is very similar to human FDP (Hoppe- Seyler's Zei
tschrift fuelPhysiologisc
he Chemie 360 , 633-7 (197).
9)), a method for measuring rat D-dimer, which is a rat-derived fibrin degradation product, has not yet been developed.
Therefore, the rat fibrin degradation product rat D-dimer, which is an important parameter of the blood coagulation and fibrinolysis-promoting state in the model animal body, is measured in the development of a thrombolytic agent, a thrombus reocclusion preventing agent, etc. using a rat thrombosis model. The need to develop methods has arisen, but the conventional techniques are ¹³¹ I and
The detection of rat thrombus using an anti-human fibrin monoclonal antibody labeled with a radioisotope such as ^99m Tc has only been studied (Thrombosi).
s Research 52 , 269-78 (198).
8), Nuclear Medicine-Commu
nications 10 , 653-9 (1989),
Immunological Celluler Bi
biology 71 , 117-24 (1993)).

[0005]

[Means for Solving the Problems] The inventors of the present invention added calcium chloride to citric acid-treated rat plasma to produce a fibrin thrombus, which was digested with plasmin, and from a mixture of the resulting degradation products, The D-dimer that mainly appears in the final fraction of the fibrin degradation process was separated. Further, this was reduced to separate into residual chains of α chain, β chain, and γ-γ chain, and among them, the γ-γ residual chain having a crosslink bond which exists only in fibrin was purified. Anti-rat D made from this
-A dimer monoclonal antibody-producing hybridoma was produced, and a monoclonal that more strongly recognizes an antigen, which is a specific degradation product of fibrin, than the rat fibrinogen antigen of 3 strains out of about 2000 cell lines obtained. A strain producing the antibody was established. As a result of extensive production and purification of anti-rat D-dimer antibody using these cell lines and repeated studies, a successful assay method by the enzyme antibody method capable of selectively quantifying rat D-dimer was successfully established.

The present invention is based on these findings and is produced by fused cells of mammalian cells and myeloma cells immunized with the γ-γ chain isolated from rat D-dimer as an antigen, and is produced rather than rat fibrinogen. Rat D
-A monoclonal antibody having a molecular weight of 74 kT, which strongly recognizes dimer, an enzyme immunoassay method for rat D-dimer characterized by using the above monoclonal antibody as an antibody, and a cell fusion strain producing the above monoclonal antibody Regarding

The rat γ-γ residual chain of rat D-dimer required for the production of the monoclonal antibody and fused cells of the present invention can be obtained as follows. First, calcium chloride was added to citrate-treated plasma of a rat to prepare a fibrin thrombus, which was digested with plasmin, subjected to Sepharose 6B gel filtration, treated with urea, and then treated with Sephadex G-.
Gel filtration of 100 is performed to separate D-dimer. Further reduction and carboxymethylation are performed, and CM-cellulose is subjected to ion exchange chromatography to obtain a γ-γ residual chain. Next, using the D-dimer γ-γ residual chain obtained above, a mammal, for example, an animal such as Balb / C mouse is immunized by a conventional method, its spleen is excised, and lymphocytes therein are purified. After that, a hybridoma producing a monoclonal antibody against D-dimer is obtained by fusing with myeloma cells cultured in advance. Fusion can be performed using polyethylene glycol, electroporation, or the like. Selection of hybridoma cells can be performed using HAT medium utilizing aminopterin resistance. Hybridomas capable of producing antibodies can be screened by an immunoblot method using D-dimer obtained from rat plasma. Other immunological techniques, such as radioimmunoassay, radioimmunoprecipitation, and enzyme immunoassay, are also used for such screening.

The present inventors have established a monoclonal antibody-producing strain that recognizes D-dimer more strongly than 3 strains of fibrinogen out of about 2000 strains by the above-mentioned screening. In addition, 4 antibody-producing strains that recognize fibrinogen and D-dimer with the same sensitivity were obtained. All of the above strains grow well even in the abdominal cavity of mice, and a large amount of antibody can be easily obtained. Rat DD / 2 of these strains
B6 and Rat DD / 28E9 have been deposited at the Institute of Biotechnology, Institute of Biotechnology, P-14995, respectively.
And the accession number of P-14996.

The antibodies produced by these strains can be highly purified by well-known purification methods such as salting out, ion exchange, and affinity chromatography using protein A or protein G.

In the enzyme immunoassay using the antibody, for example, rat D-dimer contained in a sample is immobilized, the monoclonal antibody is allowed to act on the rat D-dimer, and the amount of action is measured by the enzyme labeling method. An enzyme immunoassay for rat D-dimer characterized by the following, and an enzyme immunoassay characterized by enzyme-labeling a part of a monoclonal antibody and assaying rat D-dimer contained in a sample by a sandwich method. Embraces the law.

The antibody may be used as it is, or Fab 'or Fab obtained by digesting the antibody with pepsin or papain may be used to construct an enzyme immunoassay system. At that time, an enzyme-labeled antibody obtained by directly binding an enzyme such as alkaline phosphatase or peroxidase to the antibody may be used, or an enzyme-labeled anti-mouse IgG antibody may be used as the secondary antibody. By combining such methods, it is possible to specifically measure D-dimer in rat blood using the monoclonal antibody obtained from the culture supernatant of the D-dimer-specific monoclonal antibody-producing strain. it can. In the sandwich-type enzyme immunoassay, the monoclonal antibody is labeled with biotin, and the enzyme-labeled streptavidin is allowed to act to specifically measure rat D-dimer without crossing fibrinogen.

[0012]

The present invention will be further described with reference to the following examples.

Example 1 Calcium chloride was added to rat plasma to form a fibrin network (stabilized fibrin). Human plasmin and tissue plasminogen activator were added to decompose fibrin, and then gel filtration was performed with Sepharose 6B (Pharmacia) to give a final fraction containing D-dimer, which is the final degradation product of stabilized fibrin. Urea was added to adjust the concentration to 3 M, the pH was adjusted to 5.5, the mixture was allowed to stand at 37 ° C. for 4 hours, and then subjected to gel filtration with Sephadex G-100 (manufactured by Pharmacia). After β-mercaptoethanol was added to the eluate to cause a reaction, iodoacetic acid was added to carry out carboxymethylation, and CM-52 (manufactured by Whatman)
The ion-exchange resin was purified to obtain a γ-γ chain residual fraction having a molecular weight of 74 kilodalton (FIG. 1).

[0014]

[Figure 1]

Then, 50 μg of the γ-γ chain residue obtained by the above method was emulsified in Balb / C mice (Japan SLC) with Freund's complete adjuvant (manufactured by Difco) and then intraperitoneally administered. Two and four weeks later, the same amount of γ-γ chain residue emulsified with Freund's incomplete adjuvant (manufactured by Difco) was intraperitoneally administered. After 2 more weeks
The spleen was removed 3 days later. The spleen is a Dulbecco's prescription phosphate buffer solution (hereinafter PBS) containing no divalent ions.
After washing with (-), a ground splenocyte suspension was obtained. On the other hand, the mouse myeloma P3 × 63Ag8 ・ 653 strain to be fused with the present spleen cells is an immunoglobulin non-secretory cell derived from Balb / C mouse that has completely lost the ability to synthesize antibody-related proteins. Will be possible. This strain contains 10% fetal bovine serum (J
RPMI-1 containing RH Bioscience)
The cells were grown in 640 (Nissui Pharmaceutical Co., Ltd.) medium. 6 x 10
^Seven myeloma cells were collected by centrifugation at 1000 rpm for 10 minutes, and then mixed with splenocytes prepared from two mice. After centrifugation at 1000 rpm for 10 minutes, it was resuspended in serum-free RPMI-1640 solution. Repeat this procedure to wash the cells with 50% PEG4.
3 ml of 000 (manufactured by Lifetech Oriental Co., Ltd.) was dropped. After mixing for 1 minute, serum-free RPMI-164
30 ml of solution 0 was added dropwise and immediately centrifuged at 1000 rpm for 10 minutes. The fused cell pellet was suspended in HAT medium *. 100 μl of each suspension was added dropwise to 33 96-well microplates (Nunc). This was cultured in a 37 ° C. incubator adjusted to a carbon dioxide gas concentration of 5% for 2-3 weeks. A splenocyte-myeloma fusion cell capable of growing in HAT medium was selected.

* 100 μM hypoxanthine, 0.1 μM aminopterin, 1.6 in S-Clone medium (cloning medium; manufactured by Sanko Junyaku Co., Ltd.)
It was prepared by adding μM thymidine (all manufactured by Sigma).

The search for antibody-producing hybridomas was carried out as follows. A 96-well manifold (Biodot; manufactured by Bio-Rad) was fixed, and 1 μg of D-dimer was adsorbed on the membrane of each well. Adsorption was carried out by allowing the D-dimer solution to fall by gravity. To block the non-adsorbed portion of the membrane, 200 μl of TBS containing 1% bovine serum albumin (manufactured by Sigma) was added to each well.
Each hole was washed with TBS by the suction method. After injecting the fused cells, the culture supernatant in each hole in which cells grow in the HAT medium is transferred to each well of the manifold in an amount of 100 μ.
Allowed to react for hours. After the reaction, 0.05% of surfactant (Tw
een20; TBS (TTB) manufactured by Bio-Rad
After washing with S), an alkaline phosphatase-labeled anti-mouse IgG (manufactured by Bio-Rad) was diluted 3000 times with TBS containing 1% bovine serum albumin to obtain 100 solutions each.
μl was reacted. After the reaction, it was washed with TTBS and then 0.1
M sodium chloride, 5 mM magnesium chloride, 33 mg
/ Dl nitroblue tetrazolium, 16.5 mg /
dl Bromochloroindolyl phosphate-containing Tris-HCl buffer (pH 9.5) 100 μl of each substrate solution was dropped into each well to develop color. As a result, the hybridoma corresponding to the hole in which the deep blue pigment was deposited was picked up as an antibody-producing positive strain. By this method, 8 antibody-producing positive strains were obtained.

Example 2 Enzyme Immunoassay for Quantifying D-Dimer In order to determine the antigen specificity of the antibody recognizing the D-dimer derived from the strain obtained in Example 1, each antibody was purified to obtain immunoglobulin. It was These immunoglobulin subtypes were determined using a typing kit manufactured by Amersham. DD / 2B6 and DD / 3C11 are IgG2b and DD
/ 17A2, DD / 19D9, DD / 30A7 are Ig
M, and DD / 28E9, DD / 28F2, DD / 3
OD5 was IgG1. IgG was purified using MAbTrap ^™ GII kit manufactured by Pharmacia. 100
1.5 mg of purified IgG was obtained from ml of culture supernatant. Further, for IgM, after concentrating the culture supernatant, dialyzing against distilled water and centrifuging, the precipitate obtained was dissolved in sodium carbonate buffer and used as the antibody solution. Enzyme immunoassay was performed using these antibodies to determine the specificity. Rat D-dimer and fibrinogen were added to 0.
96-well ELISA with 100 μl in each well, serially diluted with 05M sodium carbonate buffer pH 9.6
Drop on a microplate (made by Nunc) for 4 nights
The antigen was adsorbed by storing it in. The unadsorbed portion in the plate was blocked by dropping 200 μl of PBS (−) containing 1% bovine serum albumin into each well and incubating at room temperature for 2 hours. The plate was washed well with PBS (-) containing 0.05% Tween 20 (TPBS (-)), and then purified from the culture supernatant of DD / 28E9 strain Ig.
OD ₂₈₀ = 0.0143 (10 μg /
It was diluted to a concentration of (ml) and 100 μl of this was dropped into each well. After reacting for 2 hours at room temperature, after washing with TPBS, 100 of horseradish peroxidase-labeled anti-mouse IgG (BioRad) for enzyme antibody method was used.
100 μl of 0-fold dilution was dropped into each well. A blocking solution was used as the antibody diluent. After reacting for 2 hours, TP
Washed well with BS. The substrate solution was 32.5 ml of 1 tablet of o-phenylenediamine dihydrochloride (OPD tablet; Wako Pure Chemical Industries, Ltd.) added with 7 μl of 30% hydrogen peroxide, 0.1 M citric acid / 0.2 M phosphate buffer pH 5 It was prepared by dissolving it in 0.0. Add 100 μl of this to each hole of the plate and
Color was developed for 0 minutes. 100 μl of 2N H ₂ SO ₄ solution was added to each well to stop the color reaction. Color intensity is 49
Kinetic microplate reader equipped with 0 nm filter
reader) (manufactured by Molecular Devices). In the above method, when the reaction (absorbance) with D-dimer is set to 100, DD / 28E9
Strain, DD / 30A7 strain, DD / 30D5 strain IgG and I
The response of gM to fibrinogen is 33,
38 and 15 (Table 1).

[0019]

[Table 1] Table 1 lists the subtypes of the strains obtained in Example 1 and the results of the enzyme immunoassay by the antigen solid phase method of Example 2. Numerical values in the table are D for each antibody
-Representing the cross-reactivity with fibrinogen with the reaction to dimer (A490nm) as 100%.

Next, D derived from the purified DD / 2B6 strain
-Dimers, antibodies recognizing fibrinogen and DD
The amount of D-dimer in rat plasma was quantified using an antibody that strongly recognizes D-dimer than fibrinogen derived from the / 28E9 strain. DD / 28E9 strain IgG was adjusted to 0.
OD ₂ = 0.0 using 05M Tris / HCl buffer
The antibody was adsorbed by diluting it to a concentration of 143, dropping 100 μl of the diluted solution into a 96-well ELISA microplate, and storing it at 4 ° C. overnight. The unadsorbed portion in the plate was blocked by dropping 200 μl of PBS (−) containing 1% bovine serum albumin into each well and incubating at room temperature for 2 hours. The plate was thoroughly washed with TPB (-), and then D-dimer having a known concentration was added to rat plasma as a sample and reacted for 2 hours. After reaction TPB
After thorough washing with S, DD / 2B labeled with biotin
PBS (-) containing 6% IgG containing 1% bovine serum albumin
It was diluted 500 times with 100 μl of this solution was dropped into each well. After reacting at room temperature for 2 hours, the plate was washed with TPBS, horseradish peroxidase-labeled streptavidin (manufactured by Lifetech Oriental Co., Ltd.) was diluted 2000 times with PBS (-) containing 1% bovine serum albumin, and 100 μl each It was dripped in each hole. After reacting for 90 minutes, it was thoroughly washed with TPBS. The substrate solution was subjected to color development using an OPD tablet as in the above method, and the absorbance at 490 nm was measured. Using the above method, purified rat D-dimer could be measured up to 1 μg / ml to 10 μg / ml. The result is shown in FIG.

[0021]

FIG. 2

Further, the purified D-dimer in the presence of rat plasma could be detected without crossing with plasma fibrinogen.

Next, the recognition site of the antibody produced by the hybridoma was determined by the immunoblotting method. 0.
Fibrinogen α chain, β chain at a concentration of 1 mg / ml,
50m containing 2% sodium dodecyl sulfate (SDS; Bio-Rad) glycerol and a small amount of bromphenol blue (Bio-Rad) in each solution containing D-dimer, its γ-γ residual chain, and fibrinogen degradation products
An equal amount of a sample buffer of M Tris-HCl buffer (pH 6.8) was added, and the mixture was treated in an oil bath at 100 ° C. for 5 minutes. A sample buffer containing β-mercaptoethanol was added to fibrinogen and treated in the same manner. These were electrophoresed on a 4/20% polyacrylamide gradient gel in the presence of 0.1% SDS. The protein separated on the gel was electrically transferred to a PVDF transfer membrane (manufactured by Millipore) using a transblotting apparatus (Sartblot S-II; manufactured by Sartorius). In this process, using the above-mentioned apparatus, using Trisglycine buffer containing 20% methanol, 0.25
It was conducted by energizing at 35 mA / cm ² for 35 minutes. Membrane T
After washing well with BS, the non-adsorbed part was blocked by holding in TBS containing 1% bovine serum albumin for 1 hour. Next, it was reacted with the supernatant of the cell line confirmed to have positive antibody production for 1 hour at room temperature. After the reaction, the membrane was thoroughly washed with TTBS,
After reacting with alkaline phosphatase-labeled anti-mouse IgG, color was developed with a substrate solution containing nitroblue tetrazolium and bromochloroindokul phosphate according to Example 2. The recognition site of the monoclonal antibody secreted by each cell line was determined from the developed antigen. By the above method, the DD / 28E9 strain that recognizes D-dimer, DD /
Antibodies obtained from the 30A7 strain and DD / 30D5 strains do not react with the α chain and β chain of fibrinogen but recognize the γ residual chain in the degradation products of fibrinogen and fibrin. 90-41
It was revealed that the epitope exists in the region of 2.

[0024]

INDUSTRIAL APPLICABILITY According to the present invention, cells producing a novel monoclonal antibody against rat D-dimer can be prepared. It is possible to culture it to mass-produce a D-dimer monoclonal antibody. In addition, this monoclonal antibody is more D than rat fibrinogen.
-Since it strongly recognizes dimer, it can be used to specifically and safely and conveniently measure D-dimer in rat plasma. Therefore, when developing and studying thrombolytic agents using rats as test animals. Extremely useful for

[Brief description of drawings]

FIG. 1 shows the purified γ-γ chain residual chain used as an immunizing antigen in S
6 is a photograph showing the result of DS electrophoresis.

FIG. 2 is a graph showing the results of enzyme immunoassay by the sandwich method performed in Example 2, where (□-□) is purified rat D-dimer as an antigen and (Δ-Δ) is rat fibrinogen as an antigen. Is.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12R 1:91)

Claims (7)

[Claims] 1. γ-γ isolated from rat D-dimer
A monoclonal antibody produced by a fused cell of a mammalian cell and a myeloma cell immunized with a chain as an antigen and recognizing rat D-dimer more strongly than rat fibrinogen. 2. The monoclonal antibody according to claim 1, which has a molecular weight of about 1740 kilodaltons. 3. An enzyme immunoassay method for rat D-dimer, which comprises using the monoclonal antibody according to claim 1 or 2 as an antibody. 4. The measuring method according to claim 3, which is performed by a sandwich method. 5. The rat D-dimer contained in the sample is immobilized, the monoclonal antibody according to claim 1 is allowed to act on the rat D-dimer, and the amount of action is measured by an enzyme labeling method. 3. The measuring method described in 3. 6. The method according to claim 3, wherein the monoclonal antibody is enzyme-labeled and the rat D-dimer contained in the sample is measured by the sandwich method.
The described measurement method. 7. γ-γ isolated from rat D-dimer
A cell line producing a monoclonal antibody that recognizes rat D-dimer, which is a fused cell of a mammalian cell and a myeloma cell immunized with a chain as an antigen.