CS267394B1 - Mouse lymphocytes hybridoma VÚIFN BBA6 - Google Patents

Abstract

The solution concerns a mouse lymphocytic hybridoma producing a broad-spectrum antibody against 1'human and mouse fibroblast interferon beta and against 1'human recombinant interferon alpha 2, stored in the hybridoma collection of the SAV Institute of Virology in Bratislava under the designation VÚ IFN BBA6. The monoclonal antibody of VÚ hybridoma IFN BBA6 is suitable for determining the presence of the mentioned types of interferons on the basis of inhibition of their biological activity in interferon-neutralization tests or on the basis of a specific binding reaction in immunoenzymatic tests. It is also suitable for preparative purposes for the purification of human and mouse interferon beta and human interferon alpha 2 by immunoaffinity chromatography. The monoclonal antibody can also be used for the preparation of sera specific to mouse immunoglobulin of the IgG2a subclass.

Description

The invention relates to a novel hybridoma, i. j. a hybrid one-cell organism constructed by cell fusion of the mouse myeloma line NSO and the mouse spleen lymphoid cell, producing a broad-spectrum antibody against human interferon beta, murine inteinone beta and human interferon alpha 2.

To date, antibodies to interferons have been produced by injecting each type of interferon repeatedly as an antigen into test animals, most commonly rabbits or sheep. The serum of animals thus immunized, taken after a certain period of antigen challenge, serves as a source of antibodies used primarily for quantitative or qualitative analysis of antigen - individual interferons - in research and immunodiagnostic practice. This procedure, called conventional immunization, has several drawbacks. The serum of immunized animals contains a heterogeneous mixture of antibodies, the spectrum of which is different and unrepeatable in each individual organism. As a rule, the body will produce antibodies to the desired antigen in addition to antibodies to the antigen preparation that need to be removed from sera by saturation. Consequently, batches of conventional sera are difficult to standardize 'and have a wide range of quality. For the production of each batch, a clear immunization antigen and other antigens need to be prepared to saturate the ballast antibodies against impurities.

The aforementioned drawbacks of the above-mentioned and hitherto used procedure will be eliminated if a hybridoma producing a monoclonal broad-spectrum antibody against human interferons beta, alpha 2 and against mouse interferon beta deposited in the hybridomas collection of the Institute of Virology of the SAS in Bratislava, Mlýnská dolina 1 IFN BBA6.

The hybridoma was obtained in a manner known from the literature (Köhler, G., Milstein, C .: Continuous cultures of fuse cell secreting antibody of predefined specificity. Nature, 256 (1975), 495; Gerhard, W .: Fusion of Cells in suspension and outgrowth of hybrids in conditioned medium In: Monoclonal antibodies Hybrids: A New Dimension in Biological Analyzes (RH Kennet, TJ McKearn, B. Bechtol, eds., New York, Plenum Press, (1980), 370) cloning a set of hybrid cells after fusion of the mouse myeloma line NS0 and cells obtained from the spleen of BALB / c mice immunized with a 1'-human interferon beta preparer.

The advantage of the hybridoma is that it produces a homogeneous antibody, the so-called. a non-monoclonal antibody capable of specifically reacting with 1 ' human interferons beta, alpha 2 or mouse interferon beta. Hybridoma IFN BBA6

SUBJECT YOU

The murine lymphocyte hybridoma IFN BBA6 producing a broad-spectrum anti-human IgG2a subclass rnonoclonal antibody can be cultured in vitro in animal cell media or in vivo in the peritoneal cavity of BALB / c mouse mice. From cans stored in liquid nitrogen, antibody production can be initiated without further immunization with aliquons.

Example

For the in vivo expansion of hybridoma cells, 5 χ 10 ⁶ cells were injected into the peritoneal cavity of mice. For better attachment of the cells applied, the mouse was pre-treated with paraffin oil (0.5 ml intraperitoneally) 15 days prior to transfer of the hybridoma cells. After 20 days of hybridoma growth in the peritoneal cavity, the mouse was killed and the ascitic fluid produced was collected. 7 ml of ascites fluid containing 8 mg / ml immunoglobulin was obtained. The ascites fluid containing the IFN BBA6 hybridoma product specifically inhibited the antiviral and antiproliferative activity of human fibroblast interferon beta, mouse fibroblast interferon beta, and human recombinant interferon alpha 2. The monoclonal antibody also exhibited specific binding to individual interferons in immunoenzymatic assays.

In vitro hybridoma growths as semi-suspension culture. They have a shape (rounded) and the size characteristic of myeloma cells, they contain fused cell nuclei, are aneuploid. The IFN BBA6 hybridoma cells have an ultrastructural pattern of typical myeloma cells where the predominant organelle is free and membrane bound polyribosomes. The basic culture medium is Dulbecco's modification of Eagle's minimum essential medium (Dulbecco, R. and Freetnan, G., Virology 8 (1959), 396). This medium, referred to as DM EM, is supplemented with gentamycin and inactivated horse serum (10%, Elan clone, Bratislava) for hybridoma culture. The hybridoma is cultured at 37 ° C - its mean generation time is approximately 24 hours. The antibody produced is a monoclonal immunoglobulin of IgG2a subclass.

The IFN BBA6 hybridoma can be used industrially as a source of a broad spectrum antibody to 1'human and mouse fibroblast interferon beta and against 1'human recombinant interferon alpha 2 in quantitatively or qualitatively assessing the presence of each type of interferon in the assayed material. The monoclonal antibody can also be used in the purification of individual types of interferons in immunoaffinity chromatography or as a source of a single subclass antibody (IgG2a) for the preparation of sera specific for that subclass.

Claims (2)

OBJECTIVE Mouse lymphocyte hybridoma VU IFN BBA6 producing a broad spectrum non-human IgG2a subclass 1 anti-human 2 subclass can be cultured in vitro in media suitable for animal cells, or in vivo in the peritoneal cavity of BALB / c mice. From the cans stored in liquid nitrogen, production of the antibody can be initiated without further immunization with aiitigonium. Example To propagate hybridoma cells in vivo, 5 106 106 cells were injected into the peritoneal cavity of mice. For better attachment of the applied cells, the mouse was pre-treated with paraffin oil (0.5 ml intraperitoneally) 15 days before hybridoma cell transfer. After 20 days of hybridoma growth in the peritoneal cavity, the mouse was killed and the produced ascites fluid collected. 7 ml of ascitic fluid containing 8 mg / ml immunoglobulin was obtained. Ascitic fluid containing hybridoma VU IFN BBA6 specifically inhibited the antiviral and antiproliferative activity of human fibroblast interferon beta, mouse fibroblast interferon beta and human recombinant interferon alpha 2. The monoclonal antibody also showed specific binding to individual interferons in immunoenzymatic assays. In vitro growths of hybridomas as a semi-suspension culture. They are shaped (round) and the size characteristic of myeloma cells, containing fused cell nuclei, are aneuploid. IFN BBA6 hybridoma cells have ultrastructural picture of typical myeloma cells, where free and membrane-bound polyribosomes are the predominant organelle. The basic culture medium is Dulbecco's modification of Eagle's minimal essential medium (Dulbecco, R. and Freetnan, G., Virology 8/1959 /, 396). This medium, referred to as DM EM, is supplemented with gentamycin and inactivated horse serum (10%, Elan clone, Bratislava) for hybridoma cultivation. The hybridoma is cultured at 37 ° C - its mean generation time is approximately 24 hours. The antibody produced is a monoclonal immunoglobulin of the IgG2a subclass. VU IFN BBA6 may be industrially used as a source of broad spectrum antibody to human and mouse fibroblast interferon beta and against human recombinant interferon alpha 2 in quantitative or qualitative evaluation of the presence of individual types of interferons in the investigated material. The monoclonal antibody can also be used to purify individual types of interferons in immunoaffinity chromatography or as a single subclass (IgG2a) antibody source for the production of the subclass specific sera. DISCUSSION and mouse fibroblast interferon beta and against human recombinant interferon alpha 2.