Production of IgG2 class of monoclonal antibodies against a vaccine strain of infectious bursal disease virus

An intermediate vaccine strain of IBDV was used to immunize BALB/C mice via intraperitoneal route. The spleenic lymphocytes of the mouse were fused with myeloma cells of Sp2/O/Ag-14 cell line and suspended in HAT medium for differentiation of hybrids. ELISA positive hybrids (10) were subcultured out of which 4 positive hybrids, i.e. IC2.1, IC4.1, IIB5.2 and IID2.2 were expanded. Hybrids, i.e. IC2.1 (9) and IID2.2 (2) were chosen for single cell cloning by limiting dilution technique. From these 2 hybrids, 9. clones were obtained, viz. IC2.1 (9) E9, IC2.1 (9) E11,IC2.1 (9) F11, IC2.1 (9) H9, IID2.2 (2) E10, IID2.2 (2) F7, IID2.2 (2) G7 and IID2.2 (2) G8. Four out of these clones were recorded positive for anti-IBDV antibody secretion. These 4 clones, i.e. IC2.1 (9) E9, IC2.1 (9) F11, IC2.1 (9) H9 and IID2.2 (2) G7 were injected intraperitoneally into pristane primed BALB/C mice to induce in vivo production of monoclonal antibodies. Ascitic fluid from 3 mice which were injected with clones IC2.1 (9) E9, IC2.1 (9) F11 and IID2.2 (2) G7 was collected and found to be strongly positive for the presence of anti-IBDV antibodies. The culture supernatant of 4 positive clones and ascitic fluid from 3 mice were isotyped as IgG2a using ELISA based isotyping kit.