ELISA Troubleshooting Low 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.

Problem Cause Solution
Antibody/epitope reaction problems Capture antibody failed to absorb to plate Coat the plate for longer
Use more concentrated coating components
Use Boster pre-coated ELISA Kits
Epitope recognition impeded by absorption to the plate Conjugate target protein to carrier peptide before coating to plate
Primary antibody concentration too low Increase primary antibody concentration
Incubate for longer
Loss of binding activity due to improper storage Store antibodies ant -20C or below
Avoid repeated freeze-thaw cycles
Insufficient reporter enzyme activity Enzyme inhibitor present Avoid sodium azide in HRP reactions
Avoid phosphate in AP reactions
Detection reagent old, contaminated, or wrong PH Use fresh substrate at the correct pH
Detection substrate too dilute Increase concentration of detection substrate
Incorrect incubation temperature Optimize incubation temperature
Make sure all reagents are at room temperature before beginning
Plate error Excessive washing Calibrate automatic washer to the correct pressure
Wash gently with a manual pipette
Wells dried out Cover the plate with sealing film during incubations
Well bottoms scratched by pipette tips Use caution when dispensing reagents