Inadequate Blocking of Non-Specific Binding Sites (ELISA (High Background))

Question

Accepted Answer

Uniformly elevated background across plate with high coefficient of variation. Background may be particularly high in wells with lower antigen concentrations. Common causes: 1) Blocking incubation time insufficient to saturate all binding sites 2) Blocking agent incompatible with assay system or antibodies 3) Blocking protein concentration too low 4) Blocking buffer does not match detection system species Solutions: 1) Increase blocking incubation period (typically 1-2 hours at room temperature) 2) Use 5-10% normal serum from same species as detection antibody 3) Test alternative blocking agents (BSA, casein, non-fat dry milk) 4) Ensure blocking buffer concentration is adequate (typically 3-5% protein)