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 |