DD268162B1 - PROCESS FOR THE PREPARATION OF MONOCLONAL ANTIBODIES TO HUMAN IMMUNE LOBULIN M (IGM) - Google Patents

Description

Explanation of the essence of the invention

The invention has for its object to find such monoclonal antibodies with methods that allow earliest possible selection of antibodies. The process according to the invention for the production of monoclonal antibodies against human IgM is characterized in that human IgM is isolated from the serum of healthy donors by methods known per se, such as ammonium sulphate precipitation, ultracentrifugation and ion exchange chromatography, with the female AJ 6-10 weeks old Mice are immunized by multiple intraperitoneal applications of the antigen. The lymphocytes are isolated from the spleens of the immunized mice in a known manner, fused with mouse myeloma cells of the line P3-X63-AG 8.653 and cultured the cell suspension in selection medium. The cell culture supernatants with hybrid cell growth are tested for antigen-specific antibody formation in the direct EIA. After clone selection of the positively reacting cavities and transfer into the next larger cell culture vessels, only the hybrid cells are further increased, which react in the direct EIA with human IgM, but not with the immunoglobulins of the isotypes G and A. The hybridomas obtained in this way are cloned and recloned, and the specific subclones obtained are cryoconjugated by conventional methods in liquid nitrogen.

To determine which monoclonal antibodies are suitable for binding human IgM from diluted body fluids in a capture EIA, an indirect EIA is used.

According to known methods, IgM from a diluted serum pool is bound by a polyclonal anti-human IgM solid-phase antibody and presented to the monoclonal antibodies of the cell culture supernatants as a "native antigen." The bound monoclonal antibodies are determined as in the direct NWD system There are 4 cell lines

Sifin-IgM-8D10 sifin-IgM-9G1 sifin-IgM-10F6 sifin-IgM-11G4,

0.3 in the direct and indirect EIA react equally highly active. These hybridomas have been deposited in the ZIM cell site depository under numbers ZIM-0254, ZIM-0253, ZIM-0252 and ZIM-0255.

The monoclonal antibodies of these cell lines are of the IgG, isotype. The μ-chain specificity is by the immunoblot test

busy.

The main advantage of such antibodies is the strict standardization of the production of highly specific Test reagents that are clinically relevant statements, such as the determination of the acute infection status in pregnant women (rubella, Toxoplasmosis), must be taken with the highest possible precision. The monoclonal anti-human IgM antibodies produced by the method according to the invention have the example of Antibody of the cell line Sifin-IgM-11G4 in the indirect EIA compared to the antibody of the cell line BL-IgM / 1 significantly higher Binding properties on (Figure 1). The verification of the trapping properties of the monoclonal antibody purified according to known methods Sifin-IgM-11G4, in contrast to the monoclonal antibodies BL-IgM / 1 and BL-IgM / 11, has an essential Increased sensitivity for capture EIA in the diagnosis of acute pathogen-related diseases (Figure 2). In the direct IgM antibody capture EIA can be compared to polyclonal anti-human IgM with the monoclonal antibody Sifin IgM-11G 4 for the diagnosis of acute cytomegalovirus infections (Figure 3) as well as more acute Toxoplasmosis infections significantly more sensitive test conditions are created. embodiment

1. Spleen cells of AJ mice are obtained after 3 intraperitoneal injections with a human IgM fraction and isolated according to the known techniques (Kearney et al., J. Immunol., 123 [1979], 1548) with cells of the myeloma cell line P3-X63- AG8.653 merged. The Zellsu3pension is in HAT selective medium (Littlefield, Science 145 [1964], 709) RPMI 1640 supplemented with 20% fetal calf serum, 2 mmol / l glutamine, 5 χ 10 ^"6 mol / l 2-mercaptoethanol, 2 g / l sodium bicarbonate, 10 mmol / l Hepes, 240IE / ml penicillin, 150pg / ml streptomycin, 4 x 10 ^-7 mol / l aminopterin, 1 χ 10 ^"4 mol / IHypoxanthin, 1.6 x 10" ⁶ mol / IThymidin, χ with the addition of 1 10 ⁶ mouse peritoneal sowing cells / ml culture medium at 37 ⁰ C in a water-saturated 5-6% COj atmosphere incubated.

2. For the selection of hybrid cells which secrete specific antibodies to human IgM in the culture medium, a direct EIA is performed after the first medium change. Under sterile conditions, 20 .mu.l cell culture supernatant of the master plates are transferred into 80 .mu.l buffer solution of the human IgM fraction-loaded EIA plates. The hybrid cell clones of the positive reaction cavities are transferred to the next larger tissue culture plates and their cell culture supernatants in the direct EIA are checked for the presence of specific antibodies with the coating antigens human IgG, IgA and IgM. Of the IgM-specific hybrid cells, both cryopreserved and recloned are applied.

3. To assess the suitability of the IgM-specific monoclonal antibodies for an IgM antibody capture EIA, the cell culture supernatants are checked with an indirect EIA. The antigen, human IgM from a serum pool, is presented to the monoclonal antibodies of the cell culture supernatant via an adsorptively bound polyclonal anti-human IgM antibody (goat) and the binding reaction is indicated by a peroxidase-labeled anti-mouse antibody (goat). Only 20% of the established hybridomas show a positive reaction in this test system.

4. The cell lines of the highly active in the direct and indirect test system antibodies

Sifin-IgM-8D10 (ZIM No. 0254) sifin-IgM-9G1 (ZIM No. 0253) sifin-IgM-1OF6 (ZIM No. O252) sifin-IgM-11 Q4 (ZIM No. 0255)

are propagated by the known cell culture techniques after cell transfer in Pristane (2,6,10,14-tetramethylpentadecane) treated syngeneic mice as ascites tumor.

The secreted monoclonal antibodies are purified from both the cell culture supernatant and the ascites punctate by known methods. Particularly suitable preparations can be prepared by affinity chromatography over IgM-Sepharose.

Indirect enzyme immunoassay (EIA) of monoclonal anti-human lgM antibodies (mAb) from the Cell culture supernatant. Microtest plates are coated with polyclonal anti-human IgM antibodies (SIFIN) and after binding of IgM from a human serum pool or from a serum of a myeloma patient with the indicated dilutions of Cell culture supernatants of stationary cultures of the cell lines Sifin-IgM - 11G4 and BL-IgM / 1 brought into contact. The bond of

Monoclonal anti-human IgM antibodies from the cell culture supernatants are incubated with an anti-mouse Ig antibody.

Peroxidase conjugate (SIFIN) and the substrate H] O ₂ and orthophenylenediamine indicated and evaluated photometrically.

Fig. 2

Direct enzyme immunoassay (EIA) of human IgM with monoclonal solid phase antibodies. Microplates are incubated with the saturation concentrations of the purified monoclonal antibody sifin-IgM-11 G4, BL-IgM / 1 and BL-IgM / 11 adsorbed and coated with the indicated serum concentrations of a donor pool (30 samples)

brought into contact. The binding of human IgM is monitored with a polyclonal anti-human IgM antibody

Peroxidase conjugate and the substrate H ₂ O ₂ and Orthophenyldiamin displayed and evaluated photometrically.

Fig. 3

Comparison of polyclonal and monoclonal solid phase antibodies for evaluation of cytomegalovirus-specific IgM Antibodies in positive patient sera. Microtest plates were aligned in parallel with the purified monoclonal antibody Sifin - IgM - 11G 4 and a polyclonal antibody Anti-human IgM antibody coated and selected with the indicated serum concentrations of 3 positive Patients (cytomegalovirus-specific IgM-AK) were brought into contact. The binding of pathogen-specific IgM is with a peroxidase-labeled cytomegalovirus antigen and the substrate H ₂ O ₂ and orthophenylenediamine indicated and evaluated photometrically.

Claims (1)

Claim: A method for the production of monoclonal antibodies to human immunoglobulin M by fusing splenic lymphocytes of AJ mice immunized with purified human immunoglobulin M with mouse myeloma cells of the line P3-X63-AG 8.653 and subsequent selection of the obtained cell hybrids using direct or indirect enzyme immunoassay methods and cloning, cryopreservation and culturing of the selected antibody-forming hybridomas, characterized in that the hybridomas labeled with the accession numbers ZIM 0254, ZIM 0253, ZIM 0252 or ZIM 0255 are used for antibody production. For this 3 pages drawings Field of application of the invention The invention relates to a method for the production of monoclonal antibodies to human IgM. The main application of these monoclonal antibodies is in medicine z. As in the diagnosis of acute pathogen-related diseases in clinical diagnostic institutions of the health care system. Characteristic of the known state of the art In the diagnosis of acute pathogen-related diseases, the determination of specific IgM antibodies is currently being prioritized. In addition to the classical detection methods of the separation of the patient sera by density gradient centrifugation or gel filtration and the subsequent detection of the pathogen-specific IgM by hemagglutination inhibition tests or indirect fluorescence antibody tests, enzyme immunoassay methods (EIA) have become established (Voller et al., Bull. World Health Organ. 53 [1976], 55 Blomberg et al., J.Clin Microbiol 17 (1983), 1081; Kurstak et al., Bull. World Health Organ. 64 [1986], 465). The initially selected indirect EIA methods, in which carrier-fixed antigen is specific Antibody from the patient serum binds has been pushed back in favor of direct 'IgM capture EIA' methods because of their frequent false-negative results caused by the competition of pathogen-specific IgG and IgM antibodies. In the case of the IgM antibody capture EIA, the IgM from human body fluids is selectively transferred via a carrier-fixed anti-human IgM antibody to the "fet. "Phase" (Duermeyer et al., J. Clin. Microbiol., 12, 805; Schmitz et al., J. Gon. Virol. 50 [1980], 59; Isaac et al., J. Med Virol 64 (1982), 55, Nielsen et al., J. Med. Viral 22 [1987], 67) Unrecognized IgG is removed by washing The pathogen specific detection is via a labeled antigen or via a labeled antigen-specific Antibodies have been tested against the most important infectious diseases (rubella, CMV, hepatitis A and B, toxoplasmosis, mumps, measles, syphilis, etc.) The significance of the IgM-capture EIA depends on the quality of the carrier-bound anti-body The production of conventional polyclonal antibodies requires extensive absorption procedures for the elimination of cross-reacting antibodies, and the binding characteristics sometimes vary considerably from animal to animal and decrease to decrease, so that batch standardization causes great difficulties These problems can be overcome by monoclonal anti-human / antibody (μ-chain specific). The mAbs selected for specificity and binding behavior for such a test system guarantee the standardization of the preparation of the solid phase antibody and its behavior in the test system. The monoclonal antibodies to human IgM presently available in the GDR, BL-IgM / 1 (DD-PS 230879) and BL-IgM / 11 (registered in the ZIM depository under the numbers ZIM-0056 and ZIM-0093) are for the mentioned scope of application only conditionally. In comparative studies of the toxoplasmosis and CMV EIA, the BL-IgM / 1 antibody was significantly inferior to polyclonal antibody preparations. Object of the invention The object of the invention is to provide monoclonal antibodies to human IgM in an easily controllable and cost-effective manner, which are suitable for their IgM antibody capture EIA in their reaction parameters (specificity and affinity) and ensure their stability in the "lyophilisate" range is.