CS228859B1 - Mouse lymphocytic hybridom producing an antisubstance against human erythrocytic group a-antigene - Google Patents

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusing a mouse myeloma cell 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 as anti-Aj diagnostic sera are an essential component of ABO group diagnostic sera. ABO group membership is the primary and most common serological examination in human haematological and transfusion practice. It is carried out, 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 with human saliva or animal material - such as stomach 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, immunization should be repeated. Immunization of volunteers with isolated substances may be accompanied by adverse events that can only be prevented by 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.

In addition to the desired anti-A antibodies, many other antibodies are present in the serum of any intentionally immunized or non-immunized donor whose organism is continuously exposed to a wide variety of antigenic stimuli, a mixture of unique and unrepeatable compositions, some of which directly affect the serum and erythrocyte response. 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, since the inherent nature of the present preparation of sera implies the inability to obtain two production 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 Ai and A2, possibly AiB and AzB, and in determining 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 Street No. 1083, under the designation IMG CZAS HE-02.

The 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, SC, Butcher, GW, Howard, JC: Antibodies to major histocompatibility antigens produced by hybrid cell line, Nature 266: 550, 1977) by cloning a set of hybrid cells resulting from fusion of murine myeloma cell lines P3-X63 - Ag8.653 and cells obtained from the spleen of Bald / c mice immunized with saliva of the secretor of the group specific substance A.

The lymphocyte hybridoma HE-02 produces a completely homogeneous antibody, the so-called monoclonal antibody, which specifically reacts with human type erythrocytes Ai, AiB and A2 and does not react with erythrocytes A2B, B and O. HE-02 hybridoma can be cultured under in vitro conditions in culture media suitable for animal cells or in vivo conditions in the peritoneal cavity of Balb / c mice. The HE-02 hybrid can be stored for a long time in cans stored in liquid nitrogen. Antibody production can be initiated after thawing the cell can without requiring additional antigen. The monoclonal antibody produced by the HE-02 hybridoma is specific for human erythrocyte group antigen A and is free of any ballast antibodies.

Example

In order to propagate HE-02 hybridoma cells in vivo, 3X10 6 hybridoma cells were injected into the peritoneal cavity of Balb / c mice. To improve the attachment of the applied cells and promote the development of the hybridoma in the peritoneal cavity of the mice, the animals were treated with 0.5 ml of paraffin oil injected intraperitoneally 14 days prior to the administration of the hybridoma cells. After 17 days of hybridoma growth in the peritoneal cavity, the selected mouse was sacrificed and the ascites fluid formed was collected. A total of 4.7 ml of ascites fluid containing 14 mg / ml immunoglobulin was obtained. The efficacy of the HE-02 monoclonal antibody was tested by successive two-fold dilutions of the ascitic fluid in isotonic saline in agglutination tubes according to industry standard ON number 84 3225. ⁵ , erythrocytes AiB at 1: 5 dilution XX 10 ⁴ , erythrocytes A2 at 1: 1024 dilution. Ascitic fluid did not react with erythrocytes A2B, B and O.

HE-02 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-suspension 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 (3 mM) and sodium pyruvate (1 mM). This medium (referred to as H-MEMd, Institute of Molecular Genetics of the Czechoslovak Academy of Sciences) is supplemented with penicillin, streptomycin, gentamycin, 2-mercaptoethanol (5 X 10 ^-5 M), HEPES buffer (2-4 X 10 ^-3 M) ) and inactivated bovine serum for BP (Bioveta, np, Ivanovice na Hané, 10%).

The HE-02 hybridoma is cultured at 37 ° C. The mean generation time is 16.6 h, the modal number of chromosomes 13 months after construction (fusion) is 84 chromosomes. The monoclonal antibody produced is an IgM-class immunoglobulin.

The monoclonal antibody produced by the HE-02 lymphocyte mouse hybridoma can be used in medical practice to safely distinguish human erythrocyte Aβ subclass antigen from A2B subclass antigen.

Claims (1)

SUBJECT IMG CZAS HE-02 murine lymphocyte hybridoma producing monoclonal antibody IgM class against human erythrocyte group antigen A.