CS222779B1 - Mice lymphocytar hybridom - Google Patents

Abstract

The invention relates to murine lymphocyte cells hybridoma IMGCZAS PTF-01, producing antibody to porcine transferrin subclass IgG1 deposited in the Institute's hybridoma collection of Molecular Genetics of the Czechoslovak Academy of Sciences. Hybridoma monoclonal antibody alone PTF-01 is suitable for use in radioimmunoassay and or an enzyme immunoassay.

Description

The present invention relates to a novel hybridoma, i.e., a hybrid unicellular organism, constructed by cell fusion of a murine myeloma cell line P3-K63-Ag8.653 and a mouse spleen lymphoid cell producing an antibody against porcine transferrin.

To date, anti-transferrin antibodies have been produced as well as antibodies against other plasma proteins, i.e. transferrin is injected as an antigen to test animals, most commonly rabbits, pigs or sheep. The serum of these animals, taken after some time of antigen treatment, serves as a source of antibodies used in diagnostics, particularly in the quantitative analysis of transferrin concentration in plasma or other body fluids. This procedure, called conventional immunization, has several drawbacks. The serum of immunized animals exhibits a highly heterogeneous mixture of antibodies, the spectrum of which is different and unrepeatable 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; these must be removed from sera in a complicated way, called saturation. Consequently, batches of conventional sera are difficult to standardize and are based on production in a wide quality range. This implies the need to compare results with a large number of antigen standards in conventional sera based diagnostic methods. For the production of each batch of serum, a substantially pure immunization antigen and other antigens to saturate the ballast antibodies against impurities should be prepared.

The above mentioned drawbacks of the above-mentioned procedure are eliminated if a hybridoma producing a monoclonal antibody against porcine transferrin is deposited in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská street No. 1US3 under the designation IMG OCAS PTF-O1.

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Said hybridoma was obtained by a method known in the literature [Fazekss de, St., Groth, S., Scheidigger, D .: Fraction of Raonoclonal Antibodies: Strategies and Tactics, J. Inmunol.

Meth., 35: 1-21, 1980j Galfre, G., Howe, SC, Milstein, C., Butcher, GW, Howard, JC: Antibodies to Major Histocompatibility of Antigens piDuced by Hybrid Cell Lines, Nature 266: 550, 1977 / cloning a set of hybrid cells resulting from the fusion of the mouse myeloma line P > -X? 3 / AgS.65 ' and cells obtained from the spleen of Balb / c mice immunized with porcine transferrin.

The advantage of hybridomas is that it produces a homogeneous antibody, the so-called monoclonal antibody, which is capable of specifically reacting with porcine transferrin. The PTF-01 hybridoma can be cultured xn vitro in media suitable for animal cells adapted for growth in vivo in the peritoneal cavity of the Balb / c strain. 2 cans, stored in liquid nitrogen, can be used to start antibody production without additional antigen. The antibody produced by the PTF-01 hybridoma is specific to transferrin and does not need to be rid of ballast antibodies.

Exemplary embodiment

2a, in order to multiply PTF-01 hybridoma cells in vivo and to obtain the antibody produced by it, 1.5 * 10 < 6 > hybridoma cells were applied to the peritoneal cavity of a Balb / c mouse which was irritated with 0.5 ml of paraffin oil intraperitoneally. After 18 days of hybridoma growth in the peritoneal cavity, the mouse was killed and a total of 3 ml of ascitic fluid was collected from the peritoneal cavity. The ascites fluid contained 7.7 mg / ml of immunoglobulin carrying antibody activity according to electrophoretic analysis. Antibody activity was measured by a solid phase porcine transferrin enzyme assays and a horseradish peroxidase labeled porcine anti-mouse antibody. A positive reaction was observed until the ascites fluid was diluted 1:10. The utility of the antibody produced by the PTF-02 hybridoma for tulle immunoprecipitation reactions was further tested by mixing the ascitic fluid of the PTF-01 hybridomas in equal volumes with the ascitic fluids of the PTF-02 and xTF-03 hybridomas. A mixture of these three fluids containing individual mono-3 222 779 clonal anti-porcine transferrin antibodies provided a precipitate with porcine transferrin in a double immunodiffusion in a cyan gel.

PTF-01 hybridoma cells have an ultrastructural pattern of typical myeloma cells where the predominant organelle is free and membrane-bound polyribosomes. They grow in vitro as semi-suspension cultures. The basic culture medium is Eagle's minimum essential m / dium with Hank's salt mix and supplemented with non-essential amino acids, L-glutamine (> mM), sodium pyruvate A 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 (0.05 mM) HEPES buffer (10 ml) and inactivated bovine serum for cultivation of PTF-01 hybridoma. , Ivanovice na Hané, 10%. The hybridoma is cultured at 27 ° C. The mean generation time is 18.8 hours and half a year after construction, the modal number of chromosomes was 92. The antibody produced is a monoclonal immunoglobulin of IgG1 subclass.

The specificity of the antibody was verified by cross-reaction with human, feral and ovine trans ferins by enzyme immunoassay. The response of the antibody produced by the PTF-01 hybridoma was nil. Similarly, it was verified that the antibody did not react with transferrin qysi.

A known procedure was used to construct the PTF-01 hybridoma by fusing myeloma cells with immune spleen cells under the influence of polyethylene glycol of molecular weight 1500, followed by selection in medium containing hypoxanthine, aminopterin and thymidine. In hybrid cells, these populations were repeatedly cloned in semi-liquid agar and tested for production of specific antibody ⁷ .

The PTF-01 hybridoma can be used industrially as a source of anti-porcine transferrin antibody in analytical or preparative methods. The PTF-01 hybridoma monoclonal antibody itself is suitable for use in radioimmunoassay or enzyme immunoassay.

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The method of radial immunodiffusion in the gel is preferred for its simplicity. In this method, the PTF-01 hybridoma antibody can be used in conjunction with the PTF-02 and PTF-O3 hybridoma antibodies. The precipitating lines or rings obtained in the gel are sharper than those obtained using conventional anti-swine transferrin serum.

Claims (1)

Mouse lymphocyte hybridoma IKiGizAS PTF-01 producing a monoclonal antibody of IgG1 subclass against porcine transferrin.