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Culture Supernatant Gives O.D even in the uncoated wells

Elisa Culture Supernatant

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#1 Satti

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Posted 12 June 2012 - 11:43 PM

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

#2 gebirgsziege

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Posted 13 June 2012 - 01:04 AM

did you zero your analyses?
A man cannot be too careful in the choice of his enemies. (Oscar Wilde)

#3 Ben Lomond

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Posted 13 June 2012 - 01:37 PM

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

#4 Satti

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Posted 13 June 2012 - 04:16 PM

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.

#5 Ben Lomond

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Posted 14 June 2012 - 07:47 PM

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





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