No Signal or Weak Signal in ELISA (ELISA (R&D Guide))

Question

Accepted Answer

After developing the ELISA plate, wells show no color change or very faint signal that is barely above background, even for positive controls or high-concentration standards. Common causes: 1) Reagents added in incorrect order or prepared improperly (e.g. wrong dilution factors, expired reconstituted standard) 2) Primary or secondary antibody concentration too low, or incubation time insufficient 3) Capture antibody or antigen failed to bind to plate (wrong plate type, coating time <4 h at room temperature) 4) Capture and detection antibodies recognize the same epitope in sandwich ELISA 5) Sodium azide present in buffers or antibody storage solution (inhibits HRP activity) 6) Sample analyte concentration below detection range or masked by sample matrix components Solutions: 1) Repeat experiment following exact protocol order; centrifuge lyophilized standard before reconstitution 2) Increase antibody concentration via titration or extend incubation to overnight at 4°C 3) Use ELISA-validated plates (not tissue culture plates); extend coating to overnight at 4°C 4) Verify antibodies recognize distinct epitopes; use validated matched antibody pairs 5) Remove sodium azide: increase washing steps or use azide-free buffers 6) Perform serial dilutions starting with concentrated sample; spike samples with known antigen to test for interference