CS253337B1 - Mouse lymphocytar hybridome img czas tr-14 - Google Patents

Abstract

The solution concerns mouse lymphocyte antibody-producing hybridoma reverse transcriptase deposited in the Collection of Hybridomas of the Institute of Molecular-Genetics of the CSAV under the designation RT-14. Monoclonal antibody the hybridoma RT-14 is suitable for use in enzymimmunological, radioimmunological methods and preparativnlch.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusing a mouse cell of the myeloma line Sp 2/0-Ag 14 and a mouse spleen lymphoid cell producing an antibody against reverse transcriptase.

To date, antibodies to reverse transcriptase have been prepared by repeatedly injecting reverse transcriptase as an antigen to test animals, most commonly rabbits, and the serum of such immunized animals taken some time after antigen administration serves as a source of antibodies used primarily for qualitative determination of reverse transcriptase and isolation. highly purified preparations of this enzyme. This procedure, called conventional immunization, has several disadvantages. The serum of immunized animals contains a heterogeneous mixture of antibodies, the spectrum of which varies from one organism to another and does not repeat. As a rule, the organism will produce antibodies to the impurities of the antigen preparation in addition to the antibodies to the antigen of interest; these must be removed from the sera by saturation. Consequently, batches of conventional sera are difficult to standardize and are based on production in a wide quality range. For the production of each batch, it is necessary to prepare pure immunization antigen and other antigens in order to seize ballast antibodies against impurities.

These drawbacks of the above-mentioned and previously used procedure will be eliminated if a hybridoma producing a monoclonal antibody against reverse transcriptase, deposited in the Collection of Hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská street 1083, is available IMG CZAS RT-14.

The hybridoma was obtained by a method known in the literature (Fazekas de St. Groth, S. Scheidegger, D .: Production of monoclonal antibodies. Strategies and tactics. J. Immunol. Meth., 35: 1-21, 1980; G., Howe, SC, 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 mouse myeloma cell line Sp 2 / D-Ag 14 and cells obtained from the spleen of mouse 810.A mice immunized with reverse transcriptase isolated from avian myeloblastosis virus (AMV) of 50% purity.

The advantage of the hybridoma is that it produces a homogeneous antibody, the so-called monoclonal antibody, which is capable of reacting specifically with both reverse transcriptase subunits, as detected after transfer of viral proteins (separated by reducing electrophoresis) to the nitrocellulose membrane and specific detection by indirect immunological assay (using RT-14 antibody and porcine peroxidase-conjugated porcine immunoglobulin antibody; ÚSOL, Prague). In blocking experiments, the effect of RT-14 on the enzymatic activity of reverse transcriptase was not detected.

The RT-14 hybridoma can be cultured in vitro in animal cell media and is adapted for growth in vivo in the peritoneal cavity of mouse F4 hybrids (BIO.A + BALB / c). From canned hybridoma cells stored in liquid nitrogen, antibody production can be started without additional antigen.

Example

To expand the hybridoma cells in vivo, 2x10 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) 10 days prior to transfer of the hybridoma cells. After 12 days of hybridoma growth in the peritoneal cavity, the mouse was killed and unproduced ascites fluid was collected. A total of 2.5 ml of ascites fluid containing 4 mg / ml immunoglobulin was obtained. The monoclonal antibody reacted with the specific antigen in an enzyme-immunoassay (using horseradish peroxidase-labeled pig antimouse antibody) up to a 1:10 dilution.

RT-14 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 medium with Hanks salt mixture supplemented with non-essential amino acids, L-glutamine (3 mM), sodium pyruvate (1 mM). This medium (referred to as H-MEMd, the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences) is supplemented with penicillin, streptomycin, gentamycin, 2-mercaptoestanol (0.05 nM), HEPES buffer (10 nM) and inactivated bovine serum to cultivate RT-14 hybridoma. (Bioveta, Ivanovice na Hane, 10%). The hybridoma is cultured at 37 ° C. The mean generation time is 17.8 h and 20 months after construction, the mpd chromosome count was 76. The antibody produced is a monoclonal immunoglobulin of subclass IgG 2b with kappa light chains, its isoelectric point is pH 7.1-7.5.

The monoclonal antibody produced by the RT-14 hybridoma reacts specifically with reverse transcriptase.

RT-14 can be used industrially as a source of monoclonal antibody to reverse transcriptase in analytical or preparative methods.

Claims (1)

IMG CZAS RT-14' murine lymphocyte hybridoma producing monoclonal antibody of IgG 2b subclass against avian myoblastosis reverse transcriptase.