RAPID GENERATION OF MONOCLONAL ANTIBODY-SECRETING HYBRIDOMAS AGAINST AFRICAN HORSE SICKNESS VIRUS BY INVITRO IMMUNIZATION AND THE FUSION CLONING TECHNIQUE

Splenocytes from non-immune mice were stimulated in vitro using an equ imolar mixture of factors from mixed lymphocyte reaction (MLR) and fro m phorbol-12-myristate 13-acetate (PMA) stimulated EL-4 cells, and con comitantly immunized with inactivated African horse sickness virus (AH SV) antigen serotype 4 or viral proteins 2 and 5 from AHSV serotype 9. Fusion with NSO myeloma cells was performed five days after primary o r secondary stimulation/immunization. The record of hybridoma growth a fter a standard method of fusion, expansion of cells and subsequent cl oning was compared with a fusion/cloning method in which cells were cl oned within 2 to 3 days of the fusion event. Detection of antigen spec ific antibodies in the hybridoma culture supernatants was successful o nly with cells derived from primary stimulation/immunizations. Antibod ies were detected using an indirect ELISA with the immunizing antigen coated on to the surface of the plates. Monoclonal hybridomas were iso lated within 2 to 3 weeks,using the fusion/cloning method, compared wi th the standard method, where it took 4 to 5 weeks. Although the total number of clones isolated from the fusion/cloning method was less tha n that obtained through the standard method, the yield of specific ant ibody-producing hybridomas as a percentage of the total picked was oft en more efficient with the fusion/cloning method. With respect to the immunoglobulin isotype produced, not all of the antibodies could be cl assified by the ELISA system used; 14% of anti-AHSV positive clones we re identified as IgG-secreting cells, 25% as IgM-secreting, 18% were c ross-reacting with IgG and IgM, and 43% could not be classified. Simil ar results in all aspects of the work were obtained whether a crude in fected cell extract or purified outer capsid polypeptides VP2/5, from serotype 4 and serotype 9 respectively, were used.