plates were coated with rabbit anti ige obtained from aerotic at 1 2000 dilution in bicarbonate buffer ph 9 6 with overnight incubation at 4 u00b0 c after this the plates were washed 4 times with phosphate buffered saline 0 05 tween and were incubated for 1 hour with rpmi 1640 10 fcs i e fetal calf serum at room temperature so as to saturate protein binding sites after washing dilutions of culture supernatants and of ige standards in pbs tween 0 05 were added to the plates after 2 hours of incubation plates were washed and 200 ul dilute alkaline phosphatase anti ige conjugates obtained from serotec 1 250 were added following 2 hours of incubation at room temperature the plates were washed and 200 ul of p nitrophenylphosphate in diethanolamine buffer was added the plates were incubated at 37 u00b0 c and optical densities were measured at 405 nm using an autoreader the sensitivity of this assay was 100 pg ml to perform igg and igm assays the same protocol was followed except that plates were coated with either rabbit anti human igg obtained from behring 1 2000 or rabbit anti igm obtained from behring 1 1000 and the conjugates used were conjugates of igg and alkaline phosphatase.