ELISA trouble shooting

Problem:

Test/find higher affinity primary antibody to use in the system.
Are there any compounds in the sample which can interferre with coating of antigen/antibody to the ELISA plate? (like high concentration of urea)
Has a key reagent been added?
Have some steps of the whole procedure been omitted by mistake?
Has a substrate for the enzyme been prepared correctly?
Was it a right substrate for the right enzyme?
Could a detergent concentration in the wash buffer be too high? They are usually used at 0.01-0.1 %. Remove or decrease detergent concentration in the wash buffer.
Do you have any enzyme inhibitor present in your system? Sodium azide will inhibit peroxidase activity.
Is conjugate or substrate still active? Test their activity in another system. Check expiry date of the products.
Has incubation time with the reagents been correct? Check with the manufacture recommendations.
Was the incubation temperature correct? Check if the system was not overheated (>37C) or too cold (room temperature).
Was the substrate volume correct? Check your pipet.

Non-specific binding of antibodies possible. Modify your blocking conditions.Do not apply extensive washes, since they might contribute to incresed variation by denaturation.
Plate not washed enough. Check that all the wells are actually filled with buffer during a washing step.
Conjugate concentration was too high. Check with the recommendations of the antibody manufacture.
Consider cross-reactivity of the detection antibody with coating antibody. Make a control of just coating antibody/antigen and detection antibody with no sample in between.
Too high temperature during whole procedure (>37�C).
Albumins can in some cases contribute to increased variability by causing separation of already bound proteins from medium binding surfaces.

Usually results from the not complete washing of the plate. If you titrate your samples, and wash plates manually during the whole procedure, you can start the wash from the wells with the lowest dilution of your samples.

Check that ELISA plate used was the right one for your application (there are different types of plates available).
Check if all the reagent were prepared correctly added in the right sequence.
Analyse the whole procedure with the help of the points above.

Washing problem. Wells were not completely aspirated during wash.
Plates were stuck on each other during incubations. Keep them separately if they are not rotated.
Dilution error. Check your pipets and pipeting technique.
Variable absorption of reagents to the plate. Check pH of your coating buffer, which is usually around pH 7.4 if using PBS, or pH 9.6 if using carbonate-bicarbonate buffer. Consider using another type of ELISA plates.

Recommended literature about ELISA:

"ELISA theory and practice" by J.R. Crowther, Methods in Molecular Biology TM 42 1995 Humana Press Inc ISBN 0896032795
"Immunoassays" by J.P. Gosling, Oxford University Press 2000 ISBN 0-19-963711-3
Antibody usage in the lab by L.Caponi and P.Migliorini, Springer Lab Manual 1999,
ISBN 3-540-65148-9