CS244398B1 - Mousy lymphocytoid hybridome img-czas al 01 - Google Patents

Abstract

The solution relates to mouse lymphocyte antibody-producing hybridoma human albumin deposited in the collection hybrids of the Institute of Molecular Genetics CSAV under the designation AL - 01. Monoclonal the hybridoma antibody AL-01 is suitable for use in methods of enzyme immunoassay, radioimmunological and preparative.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusing a mouse jnyeloma cell line P3-X63-Ag8.653 and a mouse spleen lymphoid cell producing an antibody against human albumin.

To date, anti-albumin antibodies have been produced by repeatedly injecting albumin as an antigen into test animals, most commonly rabbits and 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 plasma albumin and for the removal of albumin from the tested sera. This procedure, called conventional immunization, has several disadvantages. In the serum of immunized animals there is a heterogeneous mixture of antibodies whose spectrum is different and unrepeatable in each individual organism. · The organism usually generates antibodies against impurities of the antigen preparation in addition to antibodies to the desired antigen; 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 manufacture of each batch, a pure immunization antigen and other antigens to saturate the ballast antibodies against impurities should be prepared.

These drawbacks of the above-mentioned and hitherto used procedure will be eliminated if a hybridoma is available, producing a fc-f monoclonal antibody against human albumin deposited in the Collection of Hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská ul. 1083, under the designation IMG CZAS AL - 01.

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. Ifeth., 35: 1-21,180; Galfré, G., Howe, S.C., Milstein, C., jautcher, G.W., Howard, J. C. Antibodies to major histocompatibility antigens produced by hybrid cell line, Nature 266: 550, 1977 / by cloning a set of hybrid

244,398 cells resulting from the fusion of cells of the mouse myeloma line P3-X63-Ag 8.653 and cells obtained from the spleen of BALB / c mice immunized with a mixture of human serum proteins containing especially albumin and transferrin.

The advantage of the hybridoma is that it produces a homogeneous antibody, the so-called raonoclonal antibody, which is capable of specifically reacting with human albumin. The AL-01 hybridoma can be cultured in vitro in animal cell media and is adapted for growth in vivo in the peritoneal cavity of the BALB / c mouse strain. From cans stored in liquid nitrogen, antibody production can be started without additional antigen. The antibody produced by AL-01 hybridoma reacts from human serum proteins with albumin only as detected by electrophoretic transfer of serum proteins (separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate environment under reducing and non-reducing conditions) to nitrocellulose membrane and specific detection by indirect immunoassay. using AL-01 antibody and a peroxidase-conjugated pig anti-mouse immunoglobulin antibody. Antibody AL - 01 does not react with albumin in porcine, rabbit, mouse or bovine. Example:

In order to multiply the hybridoma cells in vivo, 2 x 10θ cells were injected into the peritoneal cavity. To improve attachment of the cells applied, the mouse was treated with paraffin oil (0.5 ml intraperitoneally) 13 days prior to the transfer of the hybridoma cells. After 18 days of hybridoma growth in the peritoneal cavity, the mouse was killed and unproduced ascites fluid was collected. A total of 4.6 ml of ascites fluid containing 5 mg / ml of immunoglobulin was obtained. · Monoclonal antibody reacted with specific antigen / albumin isolated by ion-exchange blood plasma chromatography on DEAE-Sepharose, pure according to SDS electrophoresis results (in enzyme-immunoassay) horseradish peroxidase / up to 1: 10 ^ ·

AL-01 hybridoma cells have an ultrastructural pattern of typical myeloma cells where the predominant organelle is free and membrane-bound polyribosomes. In vitro they grow as

- 3 244 398 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 (3mM), sodium pyruvate 1 mM / * This medium / designated H-MEMd, Institute of Molecular Genetics (for culturing AL-01 hybridoma, it is supplemented with penicillin, streptomycin, gentamycin, 2-mercapto ethanol (0.05 nM), HEPES buffer (10 rM) and inactivated bovine serum (Bioveta, Ivanovice na Hané 10%). The hybridoma is cultured at 37 ° C. The mean generation time is 15-9 hours and 13 months after construction, the modal number of chromosomes was 80. The antibody produced is monoclonal immunoglobulin of IgG1 subclass, with kappa light chains, its isoelectric point is pH 7.6 (08.0.

The monoclonal antibody produced by hybridoma AL - 01 reacts specifically with human albumin.

The AL - 01 hybridoma can be industrially used as a source of monoclonal antibody against albumin in analytical or preparative methods, particularly to remove albumin in the detection of minor serum proteins and to quantify very low albumin concentrations in urine and lung lavage.

Claims (1)

SUBJECT OF THE INVENTION Mouse lymphocyte hybridoma IMG CZAS AL-01, producing a monoclonal antibody of IgG1 subclass against human albumin.