CS265473B1 - Mouse lymphocyte hybridoma producing antibody against human chorionic gonadotropic hormone - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma producing an antibody against human choriogonadotropic hormone (HCG), which is stored in the Hybridoma Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation IMG CZAS H-3F11. The monoclonal antibodies themselves are suitable for the production of new diagnostic kits for early pregnancy diagnosis.

Description

The invention relates to novel hybridomas, i.e. hybrid cell lines which have been constructed by fusing cells of the myeloma line SP2 / O and murine spleen cells producing antibodies against human chorionicadotropic hormone (HCG). This hormone is detectable in the blood and urine of pregnant women and is completely absent in non-pregnant women. Its occurrence in the body begins after implantation of fertilized egg (about .8 days after ovulation) and the concentration of HCG then increases sharply. Diagnostic tests during pregnancy are based on evidence of HCG presence in the body.

The need for early and reliable diagnosis of pregnancy now arises especially in connection with the introduction of mini-abortions, which are tied to the first weeks after conception. With this, there is a growing demand for increased sensitivity and reliability of diagnostic tests.

Previous diagnostic tests utilize immunological reactions of conventional antisera prepared by immunizing rabbits with purified HCG. The serum of immunized animals is a source of antibodies for immunoassays. A disadvantage of the preparation of these conventional antisera is the requirement for highly purified hormone for their preparation and the individual batches of antisera differ in their activity and contain a considerably heterogeneous mixture of antibodies. HCG and other similar hormones (LH, FSH) are known to have high similarity to their alpha chain and beta chain moieties, and therefore polyvalent conventional antibodies to HCG react with these hormones. Due to a certain basal LH concentration even in non-pregnant women, conventional antibody-based agglutination tests are not able to specifically detect less than 500 IU HCG / L, and pregnancy can be detected by these antibodies only 7 to 10 days after menzes are missed.

The disadvantages of conventional antisera variability and cross-reactivity of polyvalent antibodies are eliminated by hybridoma technology.

By means of extensive screening, it is possible to select those hybridoma products - monoclonal antibodies that react only with a single antigenic determinant on the HCG beta beta subunit that is specific for the HCG molecule and which is absent on other hormone molecules. Such a monoclonal antibody is useful for the specific detection of HCG. This makes it possible to sensitize the agglutination tests hitherto used and to introduce new forms of diagnostic tests, such as the enzyme-immuno-and radio-immuno-tests.

The use of anti-HCG monoclonal antibodies allows for sensitization of HCG detection. Successful preparation of monoclonal antibodies against HCG has been described abroad and monoclonal antibodies are used for sensitive diagnosis of pregnancy. A new hybridoma producing an anti-HCG antibody is deposited in the Hybridoma Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 6, Flemingovo nám. 2 under the designation IMG CZAS H-3F11.

Said hybridoma was obtained by fusing spleen cells from BALB / c mice re-immunized with HCG with cells of the myeloma line SP2 / 0 according to a method known in the literature (Fazekas de St. Moritz).

Groth, S., Scheidigger, D .: Production of Monoclonal Antibodies: Strategies and Tactis. J. Immunol. Methods 35: 1, 1980) and cloning the obtained hybrid cell population.

The H-3F11 hybridoma can be cultured in vitro in animal cell media and adapted for growth in vivo in the peritoneal cavity of BALB / c mouse mice. H-2F11 hybridoma cells are stored in liquid nitrogen and once thawed, monoclonal antibody production can be started without further antigen requirement.

The basic culture medium for H-3F11 hybridoma cells is Eagle's minimal essential medium with Hanks salt mixture enriched with non-essential amino acids. This medium (called H-MEMd - Institute of Molecular Genetics of the Czechoslovak Academy of Sciences) is supplemented with antibiotics (penicillin, streptomycin) and 10% of inactivated calf serum for hybridoma cultivation. The hybridoma is cultured at 37 ° C and the antibodies produced are monoclonal immunoglobulins of IgG1 subclass. These antibodies react in an enzyme-linked immunosorbent assay (ELISA) with a partially purified beta subunit of HCG, with the whole HCG molecule, but do not react with non-pregnant female urinary protein preparations (containing LH, FSH and other proteins).

Preparation of monoclonal antibody

2x10 ⁶ hybridoma cells are injected into the peritoneal cavity of BALB / c mice injected with 1.0 ml paraffin oil into the peritoneal cavity 7-10 days prior to the administration of the hybridoma cells. After 10-14 days of hybridoma growth, mice develop ascitic fluid (4-6 ml / mouse) in the peritoneal cavity containing 2-4 mg antibody / ml (immunoglobulin). This can be purified from the ascites fluid by ammonium sulfate precipitation.

Antibodies present in the ascites fluid upon growth of the H-3F11 hybridoma react with HCG in an enzyme-linked immunoassay (ELISA) at a dilution of more than 1:10,000 and agglutinate HCG-bound blood cells. Thus, they can replace polyvalent antisera in previously used agglutination tests to diagnose pregnancy and can become essential reagents for the introduction of new enzyme and radioimmunoassays for early diagnosis of pregnancy.

Claims (1)

SUBJECT OF THE INVENTION Mouse lymphocyte hybridoma IMG CZAS H-3F11, producing a monoclonal antibody of IgG1 subclass against human chorionic gonadotropic hormone (HCG).