ELISA Troubleshooting Guide
Preforming enzyme-linked immunosorbent assays (ELISA) requires multiple assay components and steps, and therefore, there is often a need for troubleshooting and optimization. In this troubleshooting guide, we have listed solutions to some of the most common sources of problems for assay development.
Problem | Possible Cause | Solution |
---|---|---|
No signal or weak signal | Omission of key reagent | Check that all reagents have been added in the correct order. |
Incorrectly prepared, incomplete or wrong substrate | Make sure that the substrate selected is appropriate for the enzyme conjugate (such as pNPP for alkaline phosphatase and OPD or TMB for peroxidase; see substrate selection guide). Make sure that fresh H2O2 is added if necessary. | |
Washes too stringent | Use an automated plate washer, if available. Eliminate or reduce detergent concentration in washing buffer. | |
Incubation times inadequate | Incubation times should be appropriate for the system. Typical substrate development times vary from 10 to 30 minutes. | |
Substrate or conjugate is no longer active or is weak | Test conjugate and substrate for activity. | |
Enzyme inhibitor present | Sodium azide will inhibit peroxidase reactions. | |
Plate reader settings not optimal | Verify the wavelength and filter settings in the plate reader. | |
Incorrect assay temperature (too cold) | Use recommended incubation temperature. Bring substrates to room temperature before use. | |
Inadequate volume of substrate | Check to make sure that correct volume is delivered by pipette. | |
Blocking protein included in the coating solution | Omit blocking protein from coating solution. | |
High background | Cross-Reactivity | Detection antibody cross-reacting with coating antibody. Run appropriate controls. |
Incubation time too long | Reduce incubation time | |
Non-specific binding of antibodies | Use appropriate blocking buffer. | |
Concentration of conjugated second antibody too high | Perform dilutions to determine optimal working concentration. | |
Buffers contaminated | Use fresh buffers | |
Incorrect assay temperature | Check that the incubation temperature did not exceed 37 °C. | |
Inadequate washing | Ensure all wells are filling with wash buffer and are being aspirated completely. Use an automated plate washer, if available. | |
Contaminating enzymes present in sample | Test sample with substrate alone to check for contaminating enzyme activity. | |
Uneven color development | Incomplete washing of wells | Ensure all wells are filling with wash buffer and are being aspirated completely. Use an automated plate washer, if available. |
Poor standard curve | Wells not completely aspirated | Completely aspirate wells between steps. Use an automated plate washer, if available. |
Plates stacked during incubations | Keep plates separated if not rotating plates. | |
Pipetting error, poor dilution series | Check pipetting technique and calculations. | |
Reagents poorly mixed | Be sure that reagents are thoroughly mixed. | |
Poor or variable adsorption of reagents to plate | Check choice of coating buffer, usually PBS, pH 7.4 or carbonate-bicarbonate buffer, pH 9.6. Try extending incubating time. Consider different plates. Check homogeneity of samples. | |
Capture antibody did not bind to plate | Use proper ELISA plate; dilute in PBS without other proteins | |
Unexpected results | Omission of reagents | Be sure that reagents were prepared correctly and added in the correct order. |
Dilution error | Check pipetting technique and calculations. | |
Technique problem | Proper mixing of reagents and wash steps are critical. | |
Inappropriate ELISA plate used | If using fluorescence detection, appropriate plates must be used. | |
Poor duplicates | Uneven plate coating | Use proper ELISA plate; check coating and blocking volumes |
Insufficient or not uniform washing | Follow uniform washing procedures; check for any obstructions in washing ports. | |
Variation in incubation temperature | Follow proper procedures; avoid incubation near any heat source. |
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