CS229995B1 - Mouse lymphocyte hybridoma - Google Patents

Abstract

The invention relates to a mouse lymphocyte hybridoma producing an antibody against human erythrocyte group antigen A, deposited in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation HE-24. The monoclonal antibody of the hybridoma HE-24 alone is suitable for distinguishing human erythrocyte antigen of subgroup AiB from antigen of subgroup A2B in medical practice.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusing a mouse myeloma cell line P3-X63-Ag0.653 and a mouse spleen lymphoid cell producing an antibody against the human erythrocyte group antigen A.

Diagnostic sera against human erythrocyte group antigen A (abbreviated diagnostic anti-A sera) are an essential component of diagnostic sera sets to determine the group membership of human erythrocytes in the ABO system. Examination of group affiliation in the ABO system is the basic and most common serological examination in human haematological and transfusion practice. It is performed, for example, as an essential part of a complete pre-transfusion examination, bedside blood group control at the beginning of a blood transfusion, serological examinations in post-transfusion events, pre-natal examinations of nurses and neonates.

Anti-A diagnostic sera have so far been prepared from human blood from selected donors that have previously been shown to have naturally high levels of anti-A agglutinins or volunteer donor blood deliberately immunized with saliva of the group specific substance A or substance A prepared from human saliva or animal material - eg gastric mucosa of pigs or horses. Substances used to immunize human volunteers need to be subjected to state control of sterility, harmlessness and efficacy. Since the concentration of the desired antibodies in the serum decreases after immunization with time, it is necessary to perform immunizations repeatedly. Immunization of volunteers Isolated substances may be accompanied by adverse manifestations that can be prevented only by desensitization of immunized individuals. So far, the method used to prepare diagnosis sera of anti-A places considerable demands on material, personnel, methodological and organizational support of the production process.

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In addition to the desired anti-A antibodies, many other antibodies, some of which directly affect the reaction of serum with erythrocytes, are present in the serum of each intentionally immunized and non-immunized donor whose organism is continuously exposed to a variety of antigenic stimuli. and impair the diagnostic efficacy of the serum obtained. Such antibodies must be removed from the serum. The standardization of individual batches of diagnosis sera is difficult and always incomplete in the current method of preparation, since it is not inherent in the prior art to prepare two batches of the same characteristics. A disadvantage of the prior art method of preparing diagnostic anti-A sera is the consumption of large amounts of human blood.

In serological examinations with anti-A diagnostic sera prepared so far, erroneous results may be obtained due to the unexpected properties of both the diagnostic sera and the erythrocytes examined. Numerous difficulties and errors may occur in distinguishing subgroups A ^ and A ^, respectively. A ^ B and A? B and in the determination of weak agglutinogens A.

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Many of the problems of the prior art method of preparing diagnosis human anti-A sera as well as a number of difficulties resulting from the adverse properties of current diagnosis sera can be overcome by introducing the preparation of murine anti-A monoclonal antibodies produced by lymphocyte hybridomas.

The source of the monoclonal antibody against human erythrocyte group antigen A is a hybridoma, deposited in the hybridoma collection of the Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic in Vienna 4, 1083 under the designation IMG CZAS HE-24.

Said hybridoma was obtained by a method known in the art - Phasekas de St, Groth, S., Scheidigger, D. Production of monoclonal antibodies: Strategies and tactics, 3. Immunol. Meth, 35: 1.1980; Galfré, G., Howe, S.C., Milstein, C., Butcher, G.M., Howard

3, C.: Antibodies to major histocompatibility antigens produced by hybrid cell line. Nature, 286: 550, 1977 / cloning

229,995 a set of hybrid cells resulting from the fusion of cells of the mouse myeloma line P3-X63-Ag8,653 and cells obtained from the spleen of BALB / c mice immunized with the saliva of the secretor of the group specific substance A,

The HE-24 lymphocyte hybridoma produces a completely homogeneous antibody, the so-called monoclonal antibody, which specifically reacts with human type erythrocytes Α ^, Α ^ Β and A _2, and does not react with the A ₂ B, B and 0 ethytrocytes. cultured under in vitro conditions in animal cell culture media or in vivo conditions in the peritoneal cavity of BALB / c mouse mice. The HE-24 hybridoma can be stored in long-term preserves in liquid nitrogen. Monoclonal antibody produced by the HE-24 hybridoma is specific for the human erythrocyte group antigen A and is free of any ballast antibodies.

Example:

To propagate HE-24 hybridoma cells under in g conditions

In vitro, 1 x 10 hybridoma cells were introduced into a Legroux culture flask containing 20 ml of H »MEMd culture medium. After three days of hybridoma cell culture growth, culture medium supplemented with the monoclonal antibody HE-24 released into the medium by hybridoma cells was recovered from the flask. The potency of HE-24 monoclonal antibody was tested by successive two-fold dilutions of monoclonal antibody-containing culture medium in isotonic saline in agglutination tubes according to industry standard ON Θ4 3225. The culture medium agglutinated to ♦ / according to ON Θ4 3225 / erythrocytes A, : 1024, erythrocytes A ^ B at a 1: 512 dilution, erythrocytes A ₂ at a 1: 128 dilution. Culture media containing HE-24 monoclonal antibody did not react with erythrocytes A ₂ B, B and O.

229 995

HE-24 hybridoma cells have an ultrastructural pattern of typical myeloma cells, where the predominant organelle is free and membrane-bound polyribosomes, growing under in vitro conditions as semi-suspension cultures. The basic culture medium is Eagle's minimum essential medium with Hanks salt mixture supplemented with non-essential amino acids, L-glutamine (3mM) and sodium pyruvate (1mM). This medium (referred to as H-MEMd, OAS molecular molecular genetics) is supplemented with penicillin, streptomycin, gentamycin, 2-mercaptoethanol (5x10-^ M), HEPES buffer (2-4x10 * ^ M) and inactivated for HE-24 hybridoma culture. bovine serum for BP. (Bioveta n.p ,, Ivanovice na Hané, 10%) In order to obtain culture medium enriched for monoclonal antibody HE-24 (hybridoma cell culture supernatant), inactivated bovine serum is replaced by inactivated fetal calf serum in the culture medium.

The HE-24 hybridoma is cultured at 37 ° C, the mean generation time is 13 hours, the modal number of chromosomes 20 months after construction (fusion) is Θ6 chromosomes. The produced monoclonal antibody is a mouse IgM class immunoglobulin, lambda.

The monoclonal antibody produced by the HE-24 murine lymphocyte hybridoma can be used in medical practice to safely distinguish human erythrocyte antigen of subgroup A ^ B from antigen of subgroup A2B.

Claims (1)

SUMMARY OF THE INVENTION 229 995 Mouse lymphocyte hybrids IMG CZAS HE-24, producing a monoclonal antibody of the IgM class against human erythrocyte group antigen A.