BACKGROUND STAINING IN IMMUNOBLOT ASSAYS - REDUCTION OF SIGNAL CAUSED BY CROSS-REACTIVITY WITH BLOCKING-AGENTS

Certain serum samples produce high background in Western and direct immunoblot assays that detect human serum IgG against specific antigens. We determined that this was due to a reaction between endogenous IgG and the membrane blocking agent (we refer to this as blocking-specific background). Using milk as blocking agent, we screened 107 sera by Western immunoblot or checkerboard immunoblot assays, and found that 6.5% of sera had background intensities sufficient to interfere with the interpretation of final results. Blocking-specific background was also observed using bovine serum albumin and other animal protein-based blocking agents. As the primary antibody in these immunoblot assays was human IgG, we investigated human serum albumin as a blocking agent; this approach eliminated the problem of blocking-specific background.