High Background Signal in ELISA (ELISA (Sigma Guide))

Question

Accepted Answer

All wells including negative controls show elevated absorbance readings. Background signal obscures specific signal, reducing signal-to-noise ratio and making data interpretation difficult. Common causes: 1) Detection antibody cross-reacting with coating antibody in sandwich ELISA format 2) Non-specific binding of antibodies to plate surface due to inadequate blocking 3) Concentration of enzyme-conjugated secondary antibody too high 4) Incubation time excessive or temperature exceeding 37°C, promoting non-specific binding 5) Insufficient washing leaving unbound conjugate; wells not completely filled or aspirated during wash cycles 6) Contaminated buffers or contaminating enzymes present in sample Solutions: 1) Run appropriate controls to detect antibody cross-reactivity; select non-cross-reactive antibody pairs 2) Use appropriate blocking buffer (e.g. BSA, casein, or non-fat milk at 1-5% concentration); extend blocking time if needed 3) Perform serial dilutions of conjugated antibody to determine optimal working concentration 4) Reduce incubation time; verify incubation temperature does not exceed 37°C 5) Ensure all wells completely fill with wash buffer and are fully aspirated; use automated plate washer with verified function 6) Prepare fresh buffers; test sample alone with substrate to check for contaminating enzyme activity