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. If you don’t see your answer in this guide or would like to discuss your particular application, please contact our Technical Service Team, which is staffed by scientists with hundreds of years in combined experience in antibodies and immunoassay 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  Substrate or conjugate is no longer active or is weak Test conjugate and substrate for activity.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. Ensure all wells are filling with wash buffer and are being aspirated completely. Use an automated plate washer, if available.
  Inadequate washing Test sample with substrate alone to check for contaminating enzyme activity.
  Contaminating enzymes present in sample Ensure all wells are filling with wash buffer and are being aspirated completely.
Uneven color development Incomplete washing of wells 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.