CS265495B1 - Mouse lymphocytar hybridome producing antibodies against"core" antigen of b-hepatitis - Google Patents

Abstract

The solution relates to mouse lymphocytic hybridoma producing antibodies against "core" Hepatitis B Virus Antibody HbcAg) which is stored in the Ostavu Hybidems Collection molecular genetics of the Czechoslovak Academy of Sciences under the designation IMG CZAS HBc 13y F9C7. The antibody can be labeled horseradish peroxidase and use it for detection anti-IgM Igc antibodies in human sera, as well as for detection and affinity purification HBcAg.

Description

The invention relates to a murine lymphocyte hybridoma producing antibodies against the hepatitis B virus core antigen (HBcAg).

The hybrid cell line was constructed by fusing cells of the mouse myeloma line (SP2 / 0) and mouse spleen lymphoid cells producing antibodies to HBcAg. HBcAg is a structural component of the hepatitis B virus virion.

Determination of anti-HBcAg levels and their differentiation into IgM and IgG antibodies is essential for the diagnosis and monitoring of hepatitis B. Total antibody levels can be determined using a competition enzyme-immunoassay, where wells are coated with HBcAg and test serum is added together with monoclonal antibody labeled with horseradish peroxidase. The determination of IgM class antibodies can be performed by an enzyme-linked immunoassay, followed by the addition of test serum, HBcAg, and horseradish peroxidase labeled monoclonal antibody.

Both of these assays can be performed using a labeled monoclonal antibody produced by the hybridoma of the invention, which is deposited in the Hybridoma Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences, Prague 6, Flemingovo nam. 2 under the designation IMG CZAS HBc 13y FgC ?. Said antibodies can also be used for the detection of HBcAg and for the affinity purification of HBcAg. Said hybridoma was obtained in a manner known from the literature (Kfthler, G., Milstein, C., Nature 256:

495-497) by cloning a population of hybrid cells obtained by hybridizing cells from the spleen of immunized BALB / c mice and cells of the mouse myeloma line SP2 / 0. Mice were immunized with HBcAg purified from bacteria containing a recombinant expression plasmid. Hitherto prepared antibodies against hepatitis B virus core antigen produced by AO hybridomas 255 255,

AO ě. 258 886, have the disadvantage that there is always some loss of immunological activity when these antibodies are labeled with horseradish peroxidase. This disadvantage is avoided with the antibody produced by hybridoma HBc 13y _{g of} C ₇ F according to the invention could be labeled with horseradish peroxidase without losses occur immunological activity. This antibody can be used to detect the total level of antibodies and IgM class antibodies in human sera, to detect and to affinity purify HBcAg. The use of this antibody allows the development of a diagnostic kit for the detection of IgM class antibodies.

Hybridoma HBc 13y _{g of} C ₇ F may be cultivated in vitro in a suitable media for animal cells, and adapted to grow in vivo in the peritoneal cavity of Balb / c. From cans that are stored in liquid nitrogen, antibody production can be initiated.

Example

To amplify hybridoma cells in vivo, 1x10 ⁶ hybridoma cells were injected into the peritoneal cavity of BALB / c mouse mice. This mouse was injected with 0.7 ml paraffin oil 14 days prior to the administration of the hybridoma cells into the peritoneal cavity. After 15 days of growth in the peritoneal cavity, mice were sacrificed and unproduced ascites fluid was collected. A total of 5 ml of ascites fluid was obtained, containing an average of 5.5 mg / ml antibody. The monoclonal antibody specifically reacted with HBcAg in an enzyme immunoassay.

The basic culture medium for the hybridoma cells HBc 13y _g C ₇ F is Eagles Minimum Essential Medium with Hank's salt mixture supplemented with nonessential amino acids. This medium (referred to as the H-MEMd-State of Molecular Genetics of the Czechoslovak Academy of Sciences) is supplemented with inactivated calf serum (Bioveta, Ivanovice na Hané), penicillin and streptomycin for hybridoma culture. The hybridoma is cultured at 37 ° C. Hybridoma HBc 13y _{g of} C ₇ F produces an antibody of subclass IgG2a isoelectric point is pH 6.5 to 6.8.

A monoclonal antibody produced from clone HBc 13y _{g of} C ₇ F reacts specifically with HBcAg and the antibody can be labeled with horseradish peroxidase, but there is a loss of immunological activity. This antibody can be used to detect IgM class antibodies in human sera for the detection and affinity purification of HBcAg. This hybridoma can be industrially used as a source of anti-HBcAg antibody in diagnostic kits.

Claims (1)

SUBJECT OF THE INVENTION Mouse Lymphocyte Hybridoma IMG CZAS HBc 13y FgC? producing antibodies against hepatitis B virus core antigen