ELISA Troubleshooting -No Color Development

Sometimes there is no color development (no signal) in ELISA, and most of the case is because of carelessness as to the possible causes. Just check or verify every step to troubleshoot the unnecessary problem which can be completely avoided.

No Color
Possible causes Recommended troubleshooting
Reagents were used in the wrong order or an assay step was omitted. Check the package insert for the assay protocol and repeat the assay.
Samples were not added to diluent (indirect format only). Verify that the samples were added to the diluent.
Wrong conjugate was used, conjugate was prepared incorrectly or has deteriorated. Be sure that the conjugate used is the one that came with the kit. All conjugates are kit-and lot-specific. If preparation of a working conjugate is needed, be sure that the concentrate and diluent are mixed in correct volumes. Do not prepare the working solution too far in advance and do not save any unused portion for future use. If no conjugate preparation is necessary, be sure to pour out only the amount required for immediate use and do not return any unused portion to the stock bottle.
Incorrect or no detection antibody was added. Add appropriate detection antibody and continue.
Substrate solution was not added. Add substrate solution and continue.
Wash buffer contains sodium azide. Avoid sodium azide in the wash buffer.