Learn how to improve your ELISA assays, to help inform your decisions, optimize your experiments, and get better results. For more information download the Boster Bio ELISA Handbook.

How to resolve Weak signals in ELISA

Weak or no color development in an ELISA assay can indicate that the target protein is present in minute quantities in the sample, if at all. It can also mean that there is something wrong with the assay or the reagents that prevent efficient detection. If your control reactions indicate that an error is causing your poor results, use this troubleshooting guide to diagnose and resolve your ELISA weak signal.

What Causes Weak Signals In ELISA?

There are three main causes of weak signals in ELISA namely;

  • Antibody/epitope reaction problems
  • Insufficient reporter enzyme activity
  • Plate related errors

Antibody/Epitope Reaction Problems

Causes Solutions
Capture antibody failed to absorb to plateCoat the plate for longer
Use more concentrated coating components
Use Boster pre-coated ELISA Kits
Epitope recognition impeded by absorption to the plateConjugate target protein to carrier peptide before coating to plate
Primary antibody concentration too lowIncrease primary antibody concentration
Incubate for longer
Primary antibody concentration too lowIncrease primary antibody concentration
Incubate for longer
Loss of binding activity due to improper storageStore antibodies ant -20°C or below
Avoid repeated freeze-thaw cycles

Insufficient Reporter Enzyme Activity

Causes Solutions
Enzyme inhibitor presentAvoid sodium azide in HRP reactions
Avoid phosphate in AP reactions
Detection reagent old, contaminated, or wrong pHUse fresh substrate at the correct pH
Detection substrate too diluteIncrease the concentration of detection substrate
Incorrect incubation temperatureOptimize incubation temperature
Make sure all reagents are at room temperature before beginning

Plate Related Errors

Causes Solutions
Excessive washingCalibrate automatic washer to the correct pressure
Wash gently with a manual pipette
Wells dried outCover the plate with sealing film during incubations
Well bottoms scratched by pipette tipsUse caution when dispensing reagents