PRODUCTION OF HYBRIDOMA CELL-LINES SECRETING ANTIBODIES TO BOVINE HERPESVIRUS-1 USING PREVIOUSLY FROZEN SPLENOCYTES

Splenocytes from mice immunized with bovine herpesvirus (BHV-1) were f used as frozen and thawed or as fresh cells with mouse myeloma cells t o generate antibody-secreting hybrido,nas. The total hybridoma product ion efficiency teas 50% with 8% of them being positive for antibody se cretion in case of fusions using frozen and thawed splenocytes. Simila rly, the fusions using fresh splenocytes gave hybridoma production eff iciency of 51%, out of which 9% produced antibodies. Thus, the product ion efficiency of total number of hybridomas as well as positive hybri domas with previously frozen splenocytes was more or less equal with t hat of fresh splenocytes. Five stable positive hybridomas were subclon ed to produce monoclonals. Out of 5 monoclonal antibodies (MAbs) obtai ned, 4 MAbs (3AC3, 9AG3, 9AE3, 9CE5) were of IgG(1) subclass, whereas, the fifth MAb (9BC6) was IgG2b. Two (9AG3, 9AE3) of these MAbs weakly neutralized the viral infectivity, Thus hybridomas/monoclonals secret ing antibodies to BHV-1 could be successfully produced using previousl y frozen splenocytes. This technique enables utilisation of all spleno cytes from immunized animal and facilitates convenient fusion scheduli ng. This technique is economical and saves excess splenocytes which ot herwise have to be discarded and conserves precious antigen as mice no t used in the initial fusion need not be maintained and boosted contin ually.