CS222781B1 - Electromotor with friction brake with decreased noise - Google Patents

Abstract

The invention relates to mouse lymphocyte antibody-producing hybridoma IMGCZAS PTF-P3 IgG1 subclass against porcine transferrin, deposited in the Institute's hybridoma collection of Molecular Genetics of the Czechoslovak Academy of Sciences. Hybridoma monoclonal antibody alone PTF-03 is suitable for use in radioimmunoassays or anzymoimunoanalyee

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 F3-X6 → -Ag8.65 and a mouse spleen lymphoid cell producing an antibody against porcine transferrin.

To date, anti-transferrin antibodies are produced as well as antibodies against other plasma proteins, i.e. transferrin is injected as an antigen to test animals, most commonly to 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. In addition to the antibodies to the desired antigen, the organism will generally produce antibodies against the impurities of the antigen preparation, and these must be removed in a complicated manner, called saturation. Consequently, production batches of conventional sera are difficult to standardize and are based on production in a wide range of quality.

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 process used so far will be 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 ul.

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No. 1083 under the designation IMGOWS PTF-03.

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. I.'eth., 35: 1-21, 1960; Galfré, G., Iows, S.C., Milstein, C., Butoher,

GW, Koward, 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 mouse myeloma line P3-X63-Ag8.653 and cells obtained from the spleen of balb mice / c, immunized with porcine transferrin.

The advantage of the hybridoma is that it produces a homogeneous antibody, the so-called monoclonal antibody, which is capable of specifically reacting with porcine transferrin. The PTF-03 hybridoma can be cultured in vitro in animal cell media and is adapted for growth in vivo in the peritoneal cavity of Balb / c mice. With preserves stored in liquid nitrogen, it is possible to start producing antibody without additional antigen. The antibody produced by the hybridoma PTF-03 is specific to transferrin and does not need to be rid of ballast antibodies.

Exemplary embodiment

To multiply PTF-03 hybridoma cells in vivo and to obtain the antibody produced by it, 1.4 x 10 hybrid cells were injected into the peritoneal cavity of a Balb / c mouse, which was hybridized with 0.5 ml paraffin oil administered intraperitoneally 12 days prior to injection of the cells. . After 16 days of hybridoma growth in the peritoneal cavity, the mouse was killed and a total of 3.5 ml of ascitic fluid was collected from the peritoneal cavity. The ascites fluid contained 11 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-03 hybridoma for immunoprecipitation reactions was further tested by mixing the ascitic fluid of the PTF-05 hybridoma in the same

222,781 volumes with ascites fluids of PIF-01 and PTF-03 hybridomas.

A mixture of these three fluids containing individual monoclonal antibodies against porcine transferrin provided a precipitate with porcine transferrin in double immunodiffusion in an agar gel.

PTF-03 hybridoma cells have an ultrastructural pattern of typical myeloma cells where the predominant organelle is free and membrane-bound polyribosomes. In v; > grow as semi-supple cultures. The basic culture medium is Eagle's minimum essential medium with Hanks salt mixture and supplemented with non-essential amino acids, L-glutamine (3 πώί), sodium pyruvate (1 mil). This medium (referred to as M-LIBUd, Institute of Molecular Genetics of the Czechoslovak Academy of Sciences), is supplemented with penicillin, streptomas, gene tamy, 2-mercapto tandem (0.05 mil) with IIEPES buffer (10 mli) for culturing PTF-03 hybridomas. and inactivated bovine serum (Bioveta, Ivanovice na Hané, 10%). The hybridoma is cultured at -37 ° C. The mean generation time is 17.1 hours and half a year after construction, the model number of chromosomes was 90. 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-03 hybridoma was nil. Similarly, it was verified that the antibody did not react with transferrin to mice.

For the construction of PTF-03 hybridomas, a known method was used to fuse myeloma cells with cells from a ‘immune spleen under the influence of a polyethylene glycote of 1500 molecular weight followed by selection in a medium 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-03 hybridoma can be used industrially as a source of anti-porcine transferrin antibody in analytical or preparative methods. The monoclonal antibody of PTF-03 hybridomas alone is suitable for use in radioimmunoassay or enzyme immunoassay.

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For simplicity, the method of radial immunodiffusion in a gel is preferred. In this method, the PTP-O3 hybridoma antibody can be used in conjunction with PTP-O1 and PTF-O2 hybridoma antibodies. A mixture of these three monoclonal antibodies provides a typical irradiated precipitate with porcine trans ferrin. The precipitating lines or rings obtained in the gel are sharper than the lines and rings obtained by conventional anti-swine serum serum.

Claims (1)

OBJECT OF THE INVENTION 22Ϊ 78 Mouse lymphocyte hybridoma IUGC2AS PTF-03, producing a monoclonal antibody of IgG1 subclass against porcine transfine.