Trouble Shooting FAQ

Below is a list of many of the common example of unexpected results, along with some possible reasons what may be causing them.

1. Standards and samples developing no colour

  • Check that the kit or any of its components have not past their use by date or expired.
  • Ensure that the enzyme conjugate concentrate has been diluted correctly.
  • Only use the reagents and components that were provided with the kit and not a mixture of reagents from other kits.
  • Rule out any the possibility of incorrect reagents being added whilst the assay was being performed.
  • Carefully review assay procedure again.

2. Samples and standards developing extremely low colour

  • Make sure that the kit has not expired, checking all the individual components and making sure none have past their use by date.
  • Avoid any source of contamination, always using aseptic methods when handling reagents from bottles and vials.
  • Different pipette tips should be used for each reagent and tips must not be allowed to touch any other reagents that are already in the well.
  • Plates should be always covered at all times except during reagent addition, washing and reading stages.
  • Incorrectly diluted enzyme conjugate.
  • Wash buffer may not have been diluted correctly or poor quality of water was use in diluting the wash buffer.
  • Recommended to always use deionised water for carrying out any dilutions.
  • There is a chance that the kit may have deteriorated prematurely.
  • Need to investigate whether the kit was properly stored and that the storage insert instructions were correctly followed.

3. Samples and standards developing extreme deep blue colour

  • Check that the enzyme conjugate concentrate was not incorrectly diluted.
  • Ensure that the plate was washed properly using the correctly diluted wash buffer as per instruction manual.

4. Standard curve displaying little or no displacement

  • Avoid any source of contamination.
  • Double check that the standards have been diluted correctly.
  • Make sure the plates were correctly washed using the diluted wash buffer.
  • Make sure that the standard has not prematurely deteriorated, please contact our technical team for further investigation.

5. Duplicates displaying variability

  • Improve pipetting technique when adding reagents, in order to be more consistent, adequate and reproducible.
  • Prepare all the reagents beforehand during the assay step up stage.
  • Reagent addition steps should be all carried out in an accurate and timely manner without any interruptions.
  • Washing steps and aspiration during washing also need to reproducible, adequate and consistent.
  • Wells need to be dumped and tapped between each washing and any excess liquid present in the wells needs to be tapped out before the substrate can be added.

6. Extracted samples displaying low colour but standard curve performs correctly

  • Extracted analyte concentration is too high and needs to be diluted so it falls within the mid-range.
  • Concentrations obtained for diluted samples will also need to be multiplied by the dilution factor.
  • Extraction of the samples is inadequate and contains a solvent.
  • Carefully review assay extraction procedure recommended in the product instruction manual again.

7. Known negative samples displaying low colour development but the standard curve performs correctly

  • Eliminate interference by diluting or extracting the samples before using again.
  • Please follow the extraction procedure which is shown in the product protocol manual.

Related Pages

  1. ELISA Principle
  2. ELISA Protocol
  3. Analysing ELISA Data
  4. General ELISA FAQ
  5. ELISA Applications
  6. Comparison Between ELISA And EIA
  7. ELISA Sample Preparation
  8. Different ELISA Detection Strategies


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