3. The immune response to the antigen was assessed by measuring the titer of polyclonal antibody in mouse serum using indirect ELISA. The mice with the highest titer were splenectomized on day 3 after the last antigen injection. The splenocytes were fused with SP2/0 myeloma cells at a ratio of 5:1 using 50% (w/v) polyethylene glycol (PEG) according to the technique established by Kohler and Milstein.7 SCH772984 nmr Using this methodology, five anti-NS1 mAbs (P148.1, P148.7, P148.9, P148.L1, P148.L2) were developed and characterized. The production, purification and characterization

of the anti dengue ‘NS1 mAb’ were performed by affinity chromatography according to the published protocol.8 This purified mAb antibody was subsequently used in the ELISA assay, as the capture antibody. The bsmAb was developed by fusing two different hybridoma cell lines, P148.L1 anti-NS1 mAb and YP4 anti-HRPO mAb each hybridoma at 2 × 107 cells was separately isolated from the two cell lines VRT752271 mouse in their logarithmic growth phase. The anti-HRPO YP4 is a well-characterized rat hybridoma that was previously selected for drug resistance to 8-azaguanine,

making it sensitive to aminopterin in HAT medium. The P148.L1 (re-suspended in RPMI media, pH 7.4) was labeled with the red dye TRITC. The YP4 (re-suspended in RPMI media, pH 6.8) was labeled with the green dye FITC. Both hybridomas were incubated for 30 min in a 5% CO2 chamber (37 °C). Excess dye was removed by repeated washes (×3) with RPMI serum free media. The cells were thoroughly mixed and then centrifuged at 459× g for 7 min. The pellet was collected and suspended in RPMI. The supernatant was removed and the fusion of the two cell lines was done by drop-wise addition of 2 ml of polyethylene glycol to the cell pellet with continuous stirring for 2 min at 37 °C. The toxic effect of PEG was immediately addressed by diluting the mixture with 20 ml of serum free RPMI media. This mixture was then centrifuged at 114× g for 5 min and the cell pellet was again suspended in RPMI media containing 10% FBS. The fused cells were sorted by fluorescence-activated

cell sorting (FACS) and (-)-p-Bromotetramisole Oxalate the dual positive cells were seeded in a 96-well sterile tissue culture plate at a concentration of 1 cell/well. The cells were cultured in 20% FBS media at 37 °C with 5% CO2 and their growth was regularly monitored. Based on cell growth, after approximately two weeks of culture, the cells were screened for their activity using the bridge ELISA technique. The stable, cloned bsmAb secreting cells were seeded in a hyper flask for large-scale expansion. 7–10 days later the supernatant was harvested and centrifuged at 5000 rpm for 30 min. The collected supernatant was passed through a 0.22 μm filter to remove cell debris and the clarified supernatant was further processed to obtain pure bsmAb antibody. The purified bsmAb was then used as the detection antibody in the bsmAb ELISA immunoassay. Purified P148.