CS222780B1 - Mice lymphocytar hydridom - Google Patents

Abstract

The invention relates to mouse lymphocyte antibody-producing hybridoma IMGCZAS PTF-02 IgG1 subclass against porcine transferrin, deposited in the collection of hybrids} Institute of Molecular Genetics of the Czechoslovak Academy of Sciences. Hybridoma monoclonal antibody alone PTF-02 is suitable for use in radioimmunoassays or enEymolmunoandlyse ·

Description

(54) Mouse Lymphocyte Hybrid

The invention relates to the murine lymphocyte hybridoma IMGCZAS PTF-02, producing an antibody of the IgG1 subclass against porcine transferrin deposited in the collection of hybrids of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences.

The PTF-02 hybridoma monoclonal antibody itself is suitable for use in radioimmunoassay or enEymolmunoandlyse.

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The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by cell fusion of a murine myeloma cell line P5-X63-Ag8.653 and a murine spleen lymphoid cell producing an anti-porcine transferrin antibody.

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 the antigen has been determined, 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 produces antibodies to the Antigen of interest and antibodies to the impurities of the antigenic preparation, which must be removed from the sera in a complicated manner, called saturation. Consequently, production batches of conventional sera are difficult to standardize and are based on production over a wide quality range. This implies comparing the 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. 108 j under the designation ILíG-HAS PTF-02. .

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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., 55: 1-21 (1980); Galfré, G., Howe, S.C., Milstein, C., Sutchar,

G.I., Howard, J.C .; Produced by hybrid cell line, Naturs 266: 550, 1977 / by cloning a set of hybrid cells resulting from the fusion of murine myeloma line P5-X63 / Ag6.655 and cells derived from the spleen of Balb / c mice immunized with porcine transferrin .

An advantage of the hybridoma is that 2c produces a homogeneous antibody, the so-called monoclonal antibody, which is capable of specifically reacting with porcine transferrin. The PTF-02 hybridoma can be cultured in vitro in animal cell-appropriate m / diids and is adapted for growth in vivo in the peritoneal cavity of a 3alb / c mouse strain. 2 lconserv, stored in liquid nitrogen, can be started by producing an antibody without additional antigen. The antibody produced by the PTF-02 hybridoma is specific to transferrin and does not need to be rid of ballast antibodies.

Exemplary embodiment

For the purpose of multiplying PTF-02 hybridoma cells in vivo and obtaining the antibody produced by it, 1x10 6 hybridoma cells were applied to the peritoneal cavity of a Salb / c mouse which was irritated with 0.5 ml of paraffin oil administered intraperitoneally 7 days prior to injection of the hybridoma cells. After 24 days of hybridoma growth in the peritoneal cavity, the mouse was killed and a total of 4.5 ml of ascitic fluid was collected from the peritoneal cavity. The ascites fluid contained 10 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 µL. The usefulness of the antibody produced by the PTF-02 hybridoma for immunoprecipitation reactions has been tested longer by mixing the ascites fluid of the PTF-02 hybridoma in equal volumes with the ascites fluids of the PTF-01 and PTF-05 hybridomas. A mixture of these three fluids containing individual monoclonal antibodies against porcine transferrin provided a precipitate with porcine transferrin

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in a double immunodiffusion in an agar gel.

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Hybridoau PTF-02 cells have an ultrastructural pattern of typical nymeloma cells where the predominant organelle is free and membrane-bound polyribosomes. They grow in vitro as semi-supple cultures. The basic culture m / hole is Eagle's minimum essential medium with Hanks salt mixture and supplemented with non-essential amino acids, L-glutamine (5 mM), sodium pyruvate (1 mil). This medium (referred to as H-MEID, Institute of Molecular Genetics, Czechoslovak Academy of Sciences) is supplemented with penicillin, streptomycin, gentamycin, 2-mercaptostanol (0.05 mM) HEPES buffer m0 mtl and inactivated bovine serum for cultivation of PTF-02 hybridoma (Bioveta, Ivanovice) in Haná, 10% /. The hybridoma is cultured at 37 ° C. The mean generation time is 18.5 hours and half a year after construction, the modal number of chromosomes was 85. The antibody produced is a monoclonal immunoglobulin of IgG1 subclass.

The specificity of the antibody was verified by cross-reaction with human, horse and sheep transferrin by the enzyme immunoassay method. The antibody response produced by the PTF-02 hybridoma was nil. Similarly, it was verified that the antibody did not react with transferrin to mice.

For the construction of the PTF-02 hybridoma, a known method was used to fuse myeloma cells with immune spleen cells under the influence of polyethylene glycol of molecular weight 1500, followed by selection in m / diu containing hypoxanthine, aminopterin and thymidine. After identifying antibody-producing hybrid cell populations, these populations were repeatedly cloned in semi-liquid agar and assayed for specific antibody production.

The PTF-01 hybridoma can be used industrially as a source of anti-porcine transferrin antibody in analytical or preparative methods. The PTF-02 hybridoma monoclonal antibody itself is suitable for use in radioimmunoassay or enzyme immunoassay.

For its simplicity, the method of radial immunodiffusion in the gel is advantageous. In this method, a PTF-02 hybridoma antibody is provided

- 4,222,780 used in conjunction with PTF-01 and PTF-03 hyhridoma antibodies.

A mixture of these three monoclonal antibodies provides a typical immune precipitate with porcine transferrin. The precipitating lines or rings obtained in the gel are sharper than those obtained using conventional anti-porcine transferrin serum.

Claims (1)

SUBJECT OF THE INVENTION IMGeAS PIF-02 mouse lymphocyte hybridoma producing a monoclonal antibody of IgG1 subclass against porcine transferrin.