CS234935B1 - Mouse lymphocyte hybrid IMG CZAS B2M-01 - Google Patents

Abstract

The invention relates to a mouse lymphocyte hybridoma producing an antibody against human beta 2 microglobulin, deposited in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation B2M-01. The monoclonal antibody of the hybridoma B2M-01 itself is suitable for use in enzyme-immunological and radioimmunological analysis and for purification of beta 2 microglobulin by affinity chromatography.

Description

The invention relates to a murine lymphocyte hybridoma producing an antibody against human beta 2 microglobulin deposited in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation B2M-01. The monoclonal antibody of the hybridoma B2M-01 itself is suitable for use in enzymatic and radioimmunological assays and for purification of beta 2 microglobulin by affinity chromatography.

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The present invention relates to a novel hybridoma, i.e., a hybrid unicellular organism, constructed by fusing a mouse myeloma cell line Sp2 / O-Ag14 and a mouse spleen lymphoid cell producing an antibody against human beta 2 microglobulin.

To date, anti-beta 2 microglobulin antibodies have been produced by beta 2 microglobulin being injected repeatedly as an antigen into test animals, most commonly rabbits. The serum of such immunized animals, taken after a certain period of antigen treatment, serves as a source of antibodies used mainly for quantitative determination of beta 2 microglobulin in serum or urine by radioimmunological analysis. 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. Generally, in addition to antibodies to the desired antigen, the organism will produce antibodies against the impurities of the antigen preparation. Consequently, production batches of conventional sera are difficult to standardize and are based on production over 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 previously used procedure will be eliminated if a hybridoma producing a monoclonal antibody against beta 2 microglobulin, deposited in the Collection of Hybridoins of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská No. 1083, is available IMG CZAS B2M-01.

The 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., 35: 1-21, 1980; G., Howe, SC, Milstein, C., Butcher, GW, Hoardard, 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 cells Sp2 / O-Ag14 line and cells obtained from the spleen of Balb / c mice immunized with human beta 2 microglobulin.

The advantage of the hybridoma is that it produces a homogeneous antibody, the so-called momoclonal antibody, which is capable of specifically reacting with human beta 2 microglobulin. The B2M-01 hybridoma can be cultured in vitro in animal cell media and is adapted for growth in vivo in the peritoneal cavity of mouse strain B, alb / c. From cans stored in liquid nitrogen, antibody production can be initiated without additional antigen. The antibody produced by the B2M-01 hybridoma reacts specifically with both human beta 2 microglobulin and human cell populations carrying the beta 2 microglo4 bulin cell membrane component and does not need to be rid of ballast antibodies.

Example 1

To multiply the hybridoma cells in vivo, 5x10 ⁸ cells were injected into the peritoneal cavity of mice. To improve attachment of the cells applied, mice were treated with paraffin oil (0.5 ml intraperitoneally) 14 days prior to transfer of the hybridoma cells. After 12 days of hybridoma growth in the peritoneal cavity, the mouse was killed and the ascitic fluid produced was collected. A total of 5 ml of ascites fluid containing 2 mg / ml immunoglobulin was obtained. The antibody reacted with a specific antigen (human beta 2 microglobulin or human peripheral blood lymphocytes) in an enzyme, immunoassay (using a horseradish peroxidase labeled porcine anti-mouse antibody) up to a 1: 10 ⁶ dilution.

Example 2

A globullin fraction was prepared from ammonium sulfate precipitation from 8 ml of ascitic fluid containing antibody B2M-01 and bound to 2 ml of CNBr-activated Sepharose 4B. 1 ml of this immunosorbent was shaken for 2 hours with 10 ml of a crude solution of concentrated human urine proteins (obtained by precipitation with saturated ammonium sulfate solution from 100 ml of pathological urine). Then the immunosorbent was transferred to a column, washed 20 ml of saline and specifically adsorbed beta 2 microglobulin was washed with a total of 5 ml of 0.1 M glycine-HCl buffer pH 2.2 and the eluate was immediately neutralized with 1 M phosphate buffer pH 7.2 . The overall yield was 2.2 mg of beta 2 microglobulin, the purity of which was at least 80%.

B2M-01 hybridoma cells have an ultrastructural pattern of typical myeloma cells. Cytoplasm contains a large number of polyribosomes, mitochondria and poorly developed endoplasmic reticulum. They grow in vitro as semi-suspension cultures. The basic culture medium is Eagle's minimal 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, status of molecular genetics of the Czechoslovak Academy of Sciences) is supplemented with penicillin, streptomycin, gentamycin, 2-mercaptoethanol (0.05 mM) in HEPES buffer (10 mM) and inactivated bovine serum (Bioveta) , Ivanovice na Hané, 10%). The hybridoma is cultured at 37 ° C. The mean generation time is 16.2 hours, and 10 months after construction, the modal number of chromosomes was 92. The antibody produced is a kappa light chain monoclonal immunoglobulin of subclass IgG2a, its isoelectric point is pH 7.0.

The monoclonal antibody produced by the B2M-01 hybridoma reacts specifically with human beta 2 microglobulin.

The B2M-01 hybridoma can be used industrially as a source of monoclonal antibody against beta 2 microglobulin in analytical or preparative methods.

B2M- hybridoma monoclonal antibody

Claims (1)

SUBJECT IMG CZAS B2M-01 mouse lymphocyte hybridoma producing monoclonal antibody -01 can be used for the analytical determination of beta 2 microglobulin (in urine, serum or other body fluids) in clinical diagnosis in healthcare facilities, especially in kidney disorders. Immobilized monoclonal antibodies can be used to purify beta 2 microglobulin by affinity chromatography. to invent a subclass of IgG2a against human beta 2 microglobulin.