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

Question

Accepted Answer

No detectable signal or very weak signal across all wells, including standard wells. The plate reader shows absorbance values near baseline or significantly lower than expected. Common causes: 1) Reagents added in incorrect order or prepared incorrectly 2) Antibody concentration too low (primary or secondary) 3) Primary and secondary antibodies not compatible (species mismatch) 4) Capture antibody or antigen did not adhere to plate (wrong plate type or insufficient coating time) 5) Capture and detection antibodies recognize the same epitope in sandwich ELISA 6) Sodium azide in buffers inhibiting HRP activity Solutions: 1) Repeat experiment following protocol exactly for solution preparation and addition order 2) Increase antibody concentration through titration; extend incubation to overnight at 4°C 3) Verify secondary antibody was raised against primary antibody host species (e.g. anti-mouse for mouse primary) 4) Use ELISA-validated plates (not tissue culture plates); extend coating step to overnight at 4°C 5) Verify antibodies recognize distinct epitopes; use validated matched antibody pairs 6) Use sodium azide-free buffers or ensure sufficient washing to remove azide completely