Assay / ELISA / Binding: no signal

1. Ranked causes

   1. Detection substrate omitted, expired, or wrong for the enzymeFresh substrate on a known-positive well develops immediately.
   2. Capture/detection antibody omitted or wrong orderChecklist shows a skipped incubate; re-running with antibodies restores signal.
   3. Reader set to wrong mode/wavelength (flat zeros)Same plate reads normally after correcting λ / luminescence mode.
   4. Plate never coated / coating washed off before assayRe-coat overnight restores kit positive control.

2. Recovery steps

   1. Apply fresh substrate to a known-positive well and watch for developmentFastest split between substrate vs upstream assay failure.
   2. Confirm reader mode/wavelength matches the kitWrong readout mode presents as total zeros.
   3. Re-coat and re-run with a printed step checklist for antibody orderSkipped coating/antibody steps are common total-loss causes.

3. Controls first

   • Did kit positive-control / top standard wells also read at blank level?If yes: Substrate, antibodies, coating, or reader — not only unknowns.

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