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Culture Supernatant Gives O.D even in the uncoated wells - (Jun/13/2012 )

Dear all
I have a very basic question , What are the possible reasons due to which the culture supernatant can gives O.D even if the wells are not coated with Antigen??
How to resolve this problem .

I coat human antigen in some wells and some wells are without the antigen coating but all wells give the same results??

Is this because of blocking, Washine etc
I already check few times by changing blocking time and more vigrous washing but the result is same.

I am looking forward to have some good suggestions


did you zero your analyses?


The answer will vary depending on your assay system.

Please give more information on your ELISA procedure, including critical steps such as coating, blocking, capture and detection reagents and it may be possible to provide a useful answer

-Ben Lomond-

Coating antigen overnight at 4 centigrade diluted with autoclaved PBS only.
Block the plates with 3% BSA in 0.1% PBST for 1 hour room temperature.
Primary A.b = Culture Supernatant
Sec A.b = Anti Human IgG Alkaline phosphatase labelled
Detection Reagent = Phosphate substrate solution. 1 hour room temperature.


If you BSA is not IgG free, and your anti-human IgG is not absorbed against bovine IgG, you will get the results that you describe

Southern Biotech, Jackson, and Bethyl have bovine absorbed reagents that will work for you

good luck

-Ben Lomond-