ELISA Troubleshooting Low Signal

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ELISA Troubleshooting: Weak Signal

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 prevents 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.

Antibody/epitope reaction problemsCapture 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
Loss of binding activity due to improper storageStore antibodies ant -20C or below
Avoid repeated freeze-thaw cycles
Insufficient reporter enzyme activityEnzyme 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 concentration of detection substrate
Incorrect incubation temperatureOptimize incubation temperature
Make sure all reagents are at room temperature before beginning
Plate errorExcessive 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

Keywords:- ELISA, Troubleshooting, Weak Signal, Low Signal, Weak Staining, No Results, No signal