CS240846B1 - Mouse Lymphocytic Hybridoma IMG CZAS IN - 04 - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma, producing an antibody against porcine and human insulin, stored in the hybridoma collection of the Molecular Genetics Collection of the Czechoslovak Academy of Sciences under the designation IN - 04. The monoclonal antibody of the hybridoma IN - 04 is suitable for use in enzyme-immunological and radioimmunological analysis and for the purification of insulin by affinity chromatography.

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 porcine and human insulin.

To date, anti-insulin antibodies have been made by repeatedly injecting insulin as an antigen into test animals, most often guinea pigs. The serum of the immunized animals collected after a period of exposure to antigens serves as a source of antibodies used, in particular, for the quantitative determination of plasma insulin by radioimmunoassay. This procedure, called conventional immunization, has several disadvantages. In the serum of immunized animals there is a heterogeneous mixture of antibodies whose spectrum varies and does not repeat in each individual organism. Generally, the organism will produce antibodies to the impurities of the antigen preparation in addition to the antibodies to the desired antigens. Consequently, batches of conventional sera are difficult to standardize and are based on production in a wide quality range. For the manufacture of each batch, a pure immunization antigen and other antigens to saturate the ballast antibodies against impurities should be prepared.

The above-mentioned drawbacks of the above-mentioned and hitherto used procedure will be eliminated if a hybridoma producing a monoclonal antibody against insulin, deposited in the Collection of Hybridomas of the Institute of Molecular Genetics of the Institute of Molecular Genetics, Prague 4, Vídeňská 1083, is available IMG CZAS IN-04.

Said hybridoma was obtained by a method known in the art. (Fazekas de St. Groth, S., Scheidigger, D .: Production of Monoclonal Antibodies: Strategies and Tactics, J. Immunol. Meth., 35: 1-21, 1980; Galfré, G., Howe, S.C.,

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Milstein, C., 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 line P3-X63-Ag8.653 and cells derived from the spleen of BALÉ / c mice immunized with porcine insulin.

The advantage of the hybridoma is that it produces a homogeneous antibody, the so-called monoclonal antibody, which is capable of specifically reacting with both porcine and human insulin. The IN-04 hybridoma can be cultured in vitro in animal cell media and is adapted for growth in vivo in the peritoneal cavity of BALB / c mouse mice. From canned products stored in liquid nitrogen, antibody production can be initiated without additional antigens. The antibody produced by the hybridoma IN-04 reacts with both porcine and human insulin (i.e., recognizes a determinant common to these two very similar peptides) and does not need to be rid of ballast antibodies.

Example

To multiply the hybridoma cells in vivo, 5 x 10 6 cells were injected into the peritoneal cavity of mice. To improve attachment of the cells applied, the mouse was treated with paraffin oil (0.5 ml intraperitoneally) 14 days prior to transfer of the hybridoma cells. After 20 days of hybridoma growth in the peritoneal cavity, the mice were mice. killed and unproduced ascites fluid collected. A total of 2.0 ml of ascites fluid containing 7 mg / ml immunoglobulin was obtained. The antibody was reacted with a specific antigen (human or porcine insulin) in an enzyme immunoassay (using a horseradish peroxidase labeled porcine anti-mouse antibody) up to a 1:10 dilution. After binding of the antibody to an agarose gel, insulin could be isolated on this immunosorbent by affinity chromatography.

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IN-04 hybridoma cells have an ultrastructural pattern of typical myeloma cells. The hybridoma cells contain nuclei characterized by considerable polymorphism. Cytoplasma cells contain a large number of polyribosomes, mitochondria and poorly developed endoplasmic reticulum. They grow in vitro as semi-suspension cultures. The basic culture vessel is Eagle's minimum essential medium with Hanks salt mixture supplemented with non-essential amino acids, L-glutamine (3 mM), sodium pyruvate (1 x nM). This medium (called H-MEMd, Institute of Molecular Genetics of the Czechoslovak Academy of Sciences) is supplemented with penicillin, streptomycin, gentamycin, 2-mercaptoethanol (0.05 mM) in HEPES buffer (10 mM) and inactivated bovine serum (Bioveta, Ivanovice na Hané, 10%). The hybridoma is cultured at 37 ° C. The mean generation time is 21.5 h and 10 months after construction, the modal number of chromosomes was 89. The antibody produced is a monoclonal immunoglobulin of the light chain type IgG1 subclass, its isoelectric point is in the range of pH 7.0 - 7.2.

The monoclonal antibody produced by the hybridoma IN-04 reacts specifically with porcine and human insulin.

The IN-04 hybridoma can be used industrially as a source of anti-insulin monoclonal antibody in analytical or preparative methods.

The monoclonal antibody of hybridomas IN-04 can be used for the analytical determination of insulin in clinical diagnostics in medical facilities, particularly endocrinologically oriented. Immobilized nanoclonal antibodies can be used to purify insulin by affinity chromatography.

Claims (1)

SUBJECT OF THE INVENTION 240 848 Mouse lymphocyte hybridoma IMG CZAS IN-04, producing a monoclonal antibody of IgG1 subclass against porcine and human insulin.