title: Background staining in immunoblot assays. Reduction of signal caused by cross-reactivity with blocking agents.
abstract: 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.
authors.affiliation: Foundation for Blood Research, Scarborough, ME 04070-0190.
authors.forename: W Y
authors.initials: WY
authors.lastname: Craig
authors.forename: S E
authors.initials: SE
authors.lastname: Poulin
authors.forename: M F
authors.initials: MF
authors.lastname: Collins
authors.forename: T B
authors.initials: TB
authors.lastname: Ledue
authors.forename: R F
authors.initials: RF
authors.lastname: Ritchie
mesh_terms: D003429:Cross Reactions; D015151:Immunoblotting / Q000379:methods*; D007074:Immunoglobulin G / Q000032:analysis*; D008894:Milk Proteins; D012709:Serum Albumin / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013194:Staining and Labeling
chemical_list: D007074:Immunoglobulin G; D008894:Milk Proteins; D012709:Serum Albumin; D012710:Serum Albumin, Bovine