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Merck

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.

ProblemPossible CauseSolution
No signal or weak signalOmission of key reagentCheck that all reagents have been added in the correct order.
Incorrectly prepared, incomplete or wrong substrateMake 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 stringentUse an automated plate washer, if available. Eliminate or reduce detergent concentration in washing buffer.
Incubation times inadequateIncubation 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Substrate or conjugate is no longer active or is weakTest conjugate and substrate for activity.
Enzyme inhibitor presentSodium azide will inhibit peroxidase reactions.
Plate reader settings not optimalVerify 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 substrateCheck to make sure that correct volume is delivered by pipette.
Blocking protein included in the coating solutionOmit blocking protein from coating solution.
High backgroundCross-ReactivityDetection antibody cross-reacting with coating antibody. Run appropriate controls.
Incubation time too longReduce incubation time
Non-specific binding of antibodiesUse appropriate blocking buffer.
Concentration of conjugated second antibody too highPerform dilutions to determine optimal working concentration.
Buffers contaminatedUse fresh buffers
Incorrect assay temperatureCheck that the incubation temperature did not exceed 37 °C.
Inadequate washingEnsure all wells are filling with wash buffer and are being aspirated completely. Use an automated plate washer, if available.
Contaminating enzymes present in sampleTest sample with substrate alone to check for contaminating enzyme activity.
Uneven color developmentIncomplete washing of wellsEnsure all wells are filling with wash buffer and are being aspirated completely. Use an automated plate washer, if available.
Poor standard curveWells not completely aspiratedCompletely aspirate wells between steps. Use an automated plate washer, if available.
Plates stacked during incubationsKeep plates separated if not rotating plates.
Pipetting error, poor dilution seriesCheck pipetting technique and calculations.
Reagents poorly mixedBe sure that reagents are thoroughly mixed.
Poor or variable adsorption of reagents to plateCheck 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 plateUse proper ELISA plate; dilute in PBS without other proteins
Unexpected resultsOmission of reagentsBe sure that reagents were prepared correctly and added in the correct order.
Dilution errorCheck pipetting technique and calculations.
Technique problemProper mixing of reagents and wash steps are critical.
Inappropriate ELISA plate usedIf using fluorescence detection, appropriate plates must be used.
Poor duplicatesUneven plate coating
Use proper ELISA plate; check coating and blocking volumes
Insufficient or not uniform washingFollow 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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