mo four times at 3 week intervals at 4 day after final immunization the spleen was removed and the cells fused with nsi 1 ag4 1 myeloma cells using 50 v v polyethylene glycol gibco brl fused cell pellet was re suspended in dmem medium supplemented with 15 fbs hypoxanthine aminopterin thymidine hat medium and hybridoma cloning factor icn aurora ohio hybridoma colonies were screened for secretion of mabs that bound den infected c6 36 cells by enzyme linked immunosorbent assay elisa selected clones were subcloned by limiting dilution ascitic fluids were produced in pristane primed balb c mice hybridoma cell lines were grown in dmem medium with 10 heat inactivated fbs mabs were affinity purified by protein g sepharose 4b gel and then the activity and specificity thereof were measured by the following elisa and western blot assay for elisa assay the mabs purified in the previous step were serially diluted and added to the plates of den infected cells and incubated at room temperature for 1 hour the plates were then washed three times with pbst 0 1 and incubated with horseradish peroxidase hrp conjugated anti mouse igg jackson immunoresearch laboratories west grove pa the plates were washed five times with pbst 0 1 and incubated with the peroxidase substrate o phenylenediamine dihydrochloride.