CS233810B1 - Mousy lymphocytoid hybride generating antimatter against piggish and human insulin - Google Patents

Abstract

The invention relates to mouse lymphocytic antibody-producing hybridomas against pigs and human insulin, deposited in the Institute's hybridoma collection of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation IN-05. Monoclonal antibody alone Hybrid IN-05 is suitable for use enzymimmunological and radioimmunological analysis and for affinity affinity for insulin chromatography.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by the phase of a flask of a mouse myeloa line P3-X63-Ag8.653 and a mouse elesine lymphoid flask producing an anti-swine and human insulin antibody.

To date, anti-insulin antibodies have been produced by injecting insulin repeatedly as an antigen into test animals, most often guinea pigs. The serum of the immunized animals collected after a certain period of time of antigen treatment serves as a source of antibodies, used in particular for the quantitative determination of plasmatic insulin by radioimmunoassay. This procedure, called conventional immunization, has several disadvantages. The sera of immunized animals contain a heterogeneous mixture of antibodies, the spectrum of which varies and does not repeat in each individual organism. As a rule, the organism will produce antibodies to the impurities of the antigen preparation in addition to antibodies to the desired antigen. Consequently, production batches of conventional sera are difficult to standardize and are based on production over a wide quality range. For the production of each batch, a clear immunization antigen and other antigens are required to produce ballast antibodies against impurities.

The above drawbacks of the above-mentioned and hitherto used procedure will be eliminated if a hybrid producing a monoclonal antibody is dispersed in the insulin deposited in the Hybridoma Collection of the Institute of Molecular Genetics. ČSAV in Prague 4, Vídeňská No. 1083, under the name IK6 CZAS ΏΓ-05.

The hybrid was obtained by a method known in the literature (Fazekas de St. Groth, S., Schoidigger, D .: Production of Monoclonal Antibodies: Strategies and Tactios, J. Immunol. Meth, 35: 1-21, 1980). G., Hove, SC,

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Mlstein, C., Butoher, G. W., Howard, J.C .: Antibody of major histoompatibility antigens produced by hybrid cell llaoa, Bature 266: 550, 1977) by cloning a set of hybrid cells generated by fusion cells of mouse myeloma line P3-X63-AgG. 653 and cells obtained from the spleen of BalbZc mice immunized with porcine insulin.

The advantage of hybrids is that it produces a homogeneous antibody, the so-called monoclenal antibody, which is capable of reacting specifically with both porcine and human insulin. The hybrid »ZB-G5 can be cultured in vitro in animal cell media and adapted for growth in vivo in the peritoneal cavity of BalbZc mice. From preserves stored in liquid nitrogen, antibody production can be initiated without additional antigen. The antibody produced by the H-05 hybridoma reacts with both porcine and human insulin (i.e., it recognizes a determinant common to these two very similar peptides) and there is no need to deprive them of ballast antibodies.

Example

To multiply the hybridoma cells in vivo, 5 x θθθ cells were injected into the peritoneal cavity of mice. To improve attachment of the cells applied, the mouse was treated with paraffin oil (Θ.5 ml intraperitoneally) 14 days prior to transfer of the hybridoma cells. After 22 days of hybridoma growth in the peritoneal cavity, the mouse was killed and unproducted asiitiative fluid was collected. A total of 2.5 ml of asoitic fluid containing 11 mg / ml of immunoglobulin was obtained. The antibody 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 by affinity chromatography on this immunosorbent.

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IH-05 hybridoma cells find an ultrastructural image of typical myeloma cells. Banks will find expensive types of nuclei. The first type has a spherical shape, the second type is characterized by considerable polymorphism. Cytoplaama of both types contains a large number of polyrlbosones, nitoohondrle and weakly developed endoplasmic reticulum. They grow in vitro as semi-suspension cultures. The basic culture medium is Bagleve minimal essential medium with Haaks salt mixture supplemented with noosoneial amino acids, L-glutamine (3 ml), sodium pyruvate (1 *). This medium (known as H-MBMo, Institute of Molecular Genetics of the Czechoslovak Academy of Sciences) is supplemented with penicillin, captomtomol, gentamycin, 2-morcaptoethanel (0.05 mH) with HEPES buffer (10 mM) and inactivated bovine serum (Biovota, Ivanovice). Hané, 10%). The hybrid is cultured at 37 ° C. The mean generation Sas was 19.8 hours and 10 months after the construct, the modal number of chromosomes was 91. The antibody produced is a monoclonal immunoglobulin of IgG1 subclass with light chains! type tc, its iseelectric point is in the range of pH 6.2-6.4.

The monoclonal antibody produced by the IH-05 hybridoma reacts specifically with porcine and human insulin.

The IH-05 hybridoma can be used industrially as a source of anti-insulin monoclonal antibody in analytical or proportional methods.

The monoclonal antibody of the hybridoma IH-05 can be used for the analytical determination of insulin in clinical diagnostics in medical facilities, particularly endocrinologically oriented. The mobilized monoclonal antibody can be used to purify insulin by affinity chromatography.

Claims (1)

pSbdbžt vii / le stamp 233 810 lymphocyte hybrid DIG CZAS UJ-05, producing a monoclonal antibody of IgG1 subclass against porcine and human insulin.