I am helping a colleague establish a B cell ELISpot assay in our lab. I previously worked with T cell ELISpot without issues, so I’m a bit unsure of the current problem. We trialed using total IgG and BSA coating (as a negative control for our recombinant protein). For total IgG, we coated with unconjugated anti-human IgG, and for BSA, we used BSA in PBS. The detection antibody was the same for both—biotinylated anti-human IgG—followed by Strep-ALP and BCIP/NBT substrate. We removed the backing of the plate during washing before adding Strep-ALP, which always worked for T cell ELISpot, and followed Mabtech's recommendations for antibody concentrations.
However, when we developed the assay, a marbled pattern appeared in the wells, regardless of whether we used total IgG or BSA coating. My guesses are interference from human serum albumin in the medium or the ethanol (35%) being left on too long (as my colleague ran the experiment for the first time). Could these be the causes, or are there other factors to consider? We also checked cell viability and varied cell numbers, but the wells still showed the same pattern.
