The following are common examples of unexpected results when running Neogen's ELISA kits with possible explanations.

  1. Extreme deep blue colour development with samples and standards.
    1. The plate was not properly washed (3 x 300 µL) with the diluted wash buffer. If using an automated washer, ensure the machine is working correctly, and increase wash cycle to 5 x 300 µL.
    2. The enzyme conjugate concentrate was incorrectly diluted.

  2. Extremely low colour development with samples and standards.
    1. The wash buffer was not diluted correctly before use.
    2. A poor quality of water was used to dilute the wash buffer. Deionised water should be used for dilutions.
    3. The enzyme conjugate concentrate was incorrectly diluted.
    4. The kit prematurely deteriorated, possibly from adverse shipping and storage conditions. Investigate the condition of the kit when it was received and how it was stored prior to use. Proper storage conditions are listed in the package insert.
    5. The kit has expired. Check the expiration dates on the test kit and reagents. No component of the kit should be used past the expiration date. Do not mix any reagents or components of one kit with the reagents or components of another kit.
    6. Contamination. Always use aseptic techniques when opening and removing reagents from vials and bottles. Keep the plate covered except when adding reagents, washing or reading. Always use different pipette tips for each reagent. When pipetting do not allow the pipette tip to touch any of the reagents already in the well.

  3. No colour development with samples and standards.
    1. Improper dilution of enzyme conjugate concentrate.
    2. The kit has expired. Check the expiration dates on the test kits and reagents. No components of the kit should be used past the expiration date. Do not mix any reagents or components of one kit with the reagents or components of another kit.
    3. Incorrect addition of kit reagents. Review assay procedure.

  4. Little to no displacement with the standard curve.
    1. Incorrect dilution of standards. Refer to the dilution scheme in the package insert.
    2. Contamination.
    3. The plate was not properly washed (3 x 300 µL) with the diluted wash buffer. If using an automated washer, ensure the machine is working correctly, and increase wash cycle to 5 x 300 µL.
    4. Standard has deteriorated prematurely. Contact your Neogen representative and provide them with the kit name, lot number, expiration date and your OD readings for further investigation.

  5. The standard curve performed correctly but the known negative samples gave low colour development.
    1. Samples need to be diluted or extracted to eliminate interference. Refer to the extraction procedure in the kit insert.

  6. The standard curve performed correctly but the extracted samples gave low colour development.
    1. The concentration of the analyte in the extracted sample is too high. The extracted sample will need to be diluted before running in the assay so the sample OD reading will fit in the standard curve. When a dilution is used, the concentration determined from the standard curve must be multiplied by the dilution factor.
    2. Inadequate extraction of samples resulting in the presence of a solvent. Refer to the recommended extraction procedure in the package insert.

  7. Variability with duplicates.
    1. Inconsistent and/or inadequate pipetting technique when adding reagents. Improve pipetting technique.
    2. Inconsistent washing. The wells should be washed 3 x 300 µL with the diluted wash buffer. If using an automated washer, ensure the machine is working correctly and increase wash cycle to 5 x 300 µL.
    3. Inadequate aspiration during washing. The wells should be dumped and tapped between each washing. Tap out excess liquid in wells before adding substrate. Add substrate immediately to the wells once the excess wash buffer has been removed. If using an automated washer, ensure the machine is aspirating correctly.
    4. Interruption during assay set-up. Have all reagents prepared before assay set-up commences. Reagent addition should be performed in a timely and accurate manner.

If you experience a result that is not addressed here, please contact our Technical Service Department for assistance. 

Neogen Europe, Ltd.
The Dairy School
Auchincruive
Ayr, KA6 5HU
Scotland, UK
Tel: +44 (0) 1292 526 091
Fax: +44 (0) 1292 525 602
Email: techservice-lifesciences@neogen.com

 

Replacement/Credit Policy

Neogen will be happy to replace or issue a credit for any defective kits. We request a copy of the absorbance values, corresponding template and the lot number of the test kit in question to determine the cause of the problem.