ELISA Troubleshooting: Low Signal or No Detection

Common Causes of Low Signal

• Insufficient antigen coating: The capture antigen concentration or coating time may be too low.
• Poor antibody affinity: Antibody pairs may not recognize the same epitope or bind weakly under current buffer conditions.
• Expired or inactive enzyme conjugate: HRP or AP conjugates lose activity over time or due to improper storage.
• Incorrect substrate handling: Degraded or contaminated substrate can yield weak or no signal.
• Improper plate type: Using a low-binding plate can significantly reduce antigen adsorption efficiency.

Step-by-Step Troubleshooting Protocol

1. Verify Antigen Coating

• Check coating concentration (1–10 µg/mL for most proteins is typical).
• Ensure coating buffer pH is optimal (pH 9.6 carbonate-bicarbonate buffer is most common).
• Incubate overnight at 4°C for optimal adsorption.

2. Confirm Antibody Activity

• Test antibody binding with a positive control sample known to contain the target antigen.
• Ensure antibodies have not been repeatedly freeze-thawed.
• Optimize dilution — overly concentrated or diluted antibodies can both reduce sensitivity.

3. Check Conjugate and Substrate Integrity

• Prepare conjugates and substrates fresh whenever possible.
• Store HRP- or AP-conjugates at 4°C and avoid light exposure.
• If using colorimetric substrates (TMB, PNPP), confirm that solutions are clear and not discolored before use.

4. Evaluate Incubation Conditions

• Ensure incubation times are long enough for antigen–antibody binding (typically 1–2 hours).
• Avoid excessive washing, which can remove weakly bound antigen or antibody.
• Perform incubations at consistent temperatures (room temp or 37°C, depending on protocol).

5. Check Plate Reader and Optical Setup

• Confirm the plate reader wavelength matches your substrate’s peak absorbance (e.g., 450 nm for TMB).
• Blank the instrument using wells with substrate only.
• Ensure the plate is not scratched or contaminated, which can affect light transmission.

6. Review Sample and Matrix Factors

• Use appropriate diluents to minimize matrix interference.
• Include spiked controls to verify antigen recovery from complex matrices (e.g., serum, plasma).
• Test serial dilutions of your sample to detect the linear range of signal response.

Tips / Notes

• Use fresh coating and detection reagents whenever troubleshooting new batches.
• Run parallel wells with known positive and negative controls to benchmark signal strength.
• If using biotin–streptavidin systems, confirm that the biotinylated antibody has not degraded.
• When switching substrates (e.g., from TMB to chemiluminescent), re-optimize antibody concentrations.

Troubleshooting Table

Internal Links

• ELISA Troubleshooting: High Background
• Troubleshooting Standard Curve Issues
• Troubleshooting Color Development Problems
• Blocking Buffer Optimization Protocol