Production of Hybridoma Cell Lines Secreting Antibodies to Bovine Herpesvirus-1 Using Previously Frozen Splenocytes

Abstract

Poonam, M. and Butchaiah, G. 1998. Production of hybridoma cell lines secreting antibodies to bovine herpesvirus-1 using previously frozen splenocytes. J. Appl. Anim. Res., 14: 65–73.

Splenocytes from mice immunized with bovine herpesvirus (BHV-1) were fused as frozen and thawed or as fresh cells with mouse myeloma cells to generate antibody-secreting hybridomas. The total hybridoma production efficiency was 50% with 8% of them being positive for antibody secretion in case of fusions using frozen and thawed splenocytes. Similarly, the fusions using fresh splenocytes gave hybridoma production efficiency of 51%, out of which 9% produced antibodies. Thus, the production efficiency of total number of hybridomas as well as positive hybridomas with previously frozen splenocytes was more or less equal with that of fresh splenocytes. Five stable positive hybridomas were subcloned to produce monoclonals. Out of 5 monoclonal antibodies (MAbs) obtained, 4 MAbs (3AC3, 9AG3, 9AE3, 9CE5) were of IgG₁ subclass, whereas, the fifth MAb (9BC6) was IgG_2b. Two (9AG3, 9AE3) of these MAbs weakly neutralized the viral infectivity. Thus hybridomas/monoclonals secreting antibodies to BHV-1 could be successfully produced using previously frozen splenocytes. This technique enables utilisation of all splenocytes from immunized animal and facilitates convenient fusion scheduling. This technique is economical and saves excess splenocytes which otherwise have to be discarded and conserves precious antigen as mice not used in the initial fusion need not be maintained and boosted continually.

Key words:

• Splenocytes
• frozen
• hybridoma
• monoclonal antibody
• bovine herpesvirus-1