CS229996B1 - Mice lymphocyte hybridoma - Google Patents

Abstract

The present invention relates to a murine lymphocyte hybridoma producing an antibody against human erythrocytamine group antigen A deposited in the hydride collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under HE-18. HE-18 hybridoma monoclonal antibody alone is useful for differentiating the human A1B subtype erythrocyte antigen from the A2B subgroup in medical practice.

Description

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Lymphocyte hybrid mice

The present invention relates to a murine lymphocyte hybridoma producing an antibody against human erythrocytarn group A antigen deposited in the Hydridome Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under HE-18. The monoclonal antibody of the HE-18 hybridoma itself is useful for distinguishing human erythrocyte antigen of the A1B subgroup from the A2B subgroup antigen in medical practice.

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The present invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusing a cell of the murine myocytic line P3-X63-Ag8,653 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 used to determine the group membership of human erythrocytes in the ABO system. Examination of group membership in the ΑΘΟ 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 donated blood deliberately immunized with the 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 controls * for sterility, harmlessness and efficacy. Since the concentration of the desired antibodies in the serum decreases after immunization with time, immunization should be repeated. Immunization of volunteers with isolated substances may be accompanied by adverse events that only prevent desensitization of immunized individuals. The method used for the preparation of anti-A diagnostic sera thus far 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 serum and erythrocyte response, are present in the serum of any intentionally immunized or non-immunized donor whose organism is continuously exposed to a variety of antigenic stimuli. impair the diagnostic efficacy of the serum obtained. Such antibodies must be removed from the serum. The standardization of individual batches of diagnostic sera is difficult and always incomplete in the current method of preparation, as it is impossible to obtain two production batches of the same characteristics from the very nature of the prior art sera preparation. 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 can be manifested in distinguishing subgroups and A? A ^ B and A? B and in the determination of weak agglutinogens A.

Many of the problems of the prior art method of preparing diagnostic human anti-A sera, as well as a number of difficulties resulting from the adverse properties of current diagnostic 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 Czechoslovak Academy of Sciences in Prague 4, Vídeňská ul. 1083 under the designation IMG CZAS ΗΕ-1Θ.

Said hybridoma was obtained by a method known in the literature / Fazekas de St. Groth, S., Scheidigger, D .: Production of Monoclonal Antibodies: Strategies and Tactics, J. Immunol.Meth .:

35: 1.1980; Galfré, G., Howe, S.S., Milstein, C., Butcher, G. W., Howard, J.C .: Antibodies to Major Histocompatibility Antigens Produced by Hybrid Cell Lines. Nature, 266: 550, 1977 / by cloning

229,999 sets 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 saliva secreting the group specific substance A.

Lymphocyte hybrids HE-1B produces a completely homogeneous antibody, the so-called monoclonal antibody, which specifically reacts with human type erythrocytes A ^, A ^ B and A ^ and does not react with erythrocytes A ₂ B, B and 0. Hybridoma ΗΕ-1Θ is possible 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-18 hybridoma can be stored in long-term preserves in liquid nitrogen. Antibody production can be initiated after thawing the cell canned without the need for an additional antigen. The monoclonal antibody produced by the HE-18 hybridoma is specific for human erythrocyte group antigen A and is free of any ballast antibodies.

Example:

To propagate HE-18 hybridoma cells under g conditions

in vitro, 1x10 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-18 released into the medium by hybridoma cells was recovered from the flask. The efficacy of HE-18 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 84 3225. The culture medium agglutinated to / according to UN 84 3225 / erythrocytes A ^ at 1: 512 , N erythrocytes in a dilution 1: 256, A ₂ erythrocytes in a dilution 1:64. Culture medium containing HE-18 monoclonal antibody did not react with erythrocytes A ₂ B, B and O.

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HE-18 hybridoma cells have an ultrastructural pattern of typical myeloma cells where the predominant organelle is free and membrane-bound polyribosomes. They grow in the form of semi-suspending cultures under in vitro conditions. 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 Z1mM). This medium (referred to as H-MEMd, OAS molecular molecular genetics) is supplemented with penicillin, streptomycin, gentamycin, 2-mercaptoethanol (5 x 10 5 | »|), HEPES buffer (2-4x10 * ^ I) to cultivate HE-18 hybridoma. * l / and inactivated bovine serum for BP (Bioveta np, Ivanovice na Hané, 10%). In order to obtain culture medium enriched for monoclonal antibody HE-18 (hybridoma cell culture supernatant) for in vitro diagnostic purposes, inactivated bovirus serum is replaced by inactivated fatal calf serum in the culture medium.

The HE-18 hybridoma is cultured at 37 ° C. The mean generation time is 17 hours, the modifying number of chromosomes 20 months after construction (fusion) is 87 chromosomes. The monoclonal antibody produced is an IgM class immunoglobulin,

The monoclonal antibody produced by the HE-18 murine lymphocyte hybridoma can be used in medical practice to safely distinguish the human erythrocyte antigen of subgroup A, jB from the antigen of subgroup A2B.

Claims (1)

OBJECT OF THE INVENTION 229 996 Mouse lymphocyte hybridoma IMG CZAS HE-18, producing a monoclonal antibody of the IgM class against human erythrocyte group antigen A.