High Uniform Background Signal (ELISA (R&D Guide))

Question

Accepted Answer

All wells show elevated absorbance including blanks and negative controls, creating a uniformly high background that reduces signal-to-noise ratio and compresses the standard curve. Common causes: 1) Insufficient washing (residual unbound antibody/HRP) or inadequate blocking of plate surface 2) Primary or secondary antibody concentration too high 3) TMB substrate solution prepared too early and turned blue before addition, or plate read after delay 4) HRP-conjugate concentration too high 5) HRP contamination in reused plastics (reservoirs, plate sealers, pipette tips) or buffers Solutions: 1) Increase wash cycles and/or duration; add 30-second soak between washes; add 0.01-0.1% Tween-20 to wash buffer 2) Increase blocking time or blocker concentration (BSA, casein, or gelatin) 3) Decrease primary/secondary antibody concentration through titration 4) Mix TMB substrate immediately before adding to plate; read plate immediately after stop solution 5) Use fresh plastics for each step; prepare fresh buffers; check and adjust HRP-conjugate dilution