Assay / ELISA / Binding: low signal

1. Ranked causes

   1. Primary/capture antibody concentration too low or degraded (lot / storage)Even top standard-curve points read low.
   2. Coating step incomplete (short time or wrong buffer pH)Signal weak across the whole plate, not sample-specific.
   3. Sample degraded or diluted past the assay's dynamic rangeOnly sample wells are low; standards read normally.
   4. Substrate stopped too earlyColor visibly still developing at the read timestamp.

2. Recovery steps

   1. Increase primary/capture antibody concentration or use a fresh aliquotConfirms whether the reagent itself has lost activity.
   2. Re-coat with fresh carbonate/bicarbonate buffer, pH 9.6, overnight at 4°CCoating efficiency is the most common silent failure in ELISA.
   3. Let substrate develop 5–10 more minutes before stoppingUnder-development is easy to miss and easy to fix.
   4. Re-run samples at a less dilute concentrationPulls the signal back inside the standard curve's dynamic range.

3. Controls first

   • Did the top standard-curve / positive-control wells also read low?If yes: Assay-wide chemistry problem (coating/antibody/substrate), not a sample issue.

Diagnose your run

Opens Diagnostics capture with this first-party playbook behind it. Sign in if the workspace asks.