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What antibodies to use for ELISA??


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#1 alexanam

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Posted 01 September 2009 - 03:15 PM

Hello,
What types of antibodies does everyone use for sandwich ELISA??
Coat with a monoclonal, then detect with a polyclonal or visa versa?
Can you use abs from the same animal? For example, rabbit Pab for coating AND for detection?
Any thoughts or suggestions and what has worked for you would be most appreciated!
Thank you!

#2 Bio Doc

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Posted 02 September 2009 - 05:04 AM

Hello,
What types of antibodies does everyone use for sandwich ELISA??
Coat with a monoclonal, then detect with a polyclonal or visa versa?
Can you use abs from the same animal? For example, rabbit Pab for coating AND for detection?
Any thoughts or suggestions and what has worked for you would be most appreciated!
Thank you!


The short answer: you can use either polyclonal or mAbs for sandwich ELISAs. HOWEVER, you need to be careful as to have the proper controls. Additionally, you theoretically can use Abs from the same animal, but I have never done that (mustly because I've never had too). Again, all comes down to proper controls and blocking non-specific binding.

More details: As for mAb as the primary capture vs. polyclonal Abs, I have done both with success. Just make sure you have control wells to substract out non-specific signals (one well without primary capture but everything else, one well with primary capture Ab but without the secondary capture antibody, and one well with just the secondary capture antibody). At the end, you'll only be limited by the non-specific signals so I hope you are working with robust capture antibodies (either mAb or polyclonal sources). Whenever possible, use as pure of an antigen as you can (helps eliminate the issue of cross reactivity or specificity issues and background signals). If you are capturing crude preparations, expect higher backgrounds and some difficulties in answering how specific your antibodies truly are.

Hope my thoughts help.

-Bio Doc

#3 sgt4boston

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Posted 02 September 2009 - 12:05 PM

The ag cross reactivity or specificity will be a property of the ab and not the purity of the ag used in the assay. You can determine the cross reactivity during your screening process of your animal bleeds or cell culture supernatants or when you run your dose response curve construct curves using the structurally similar compounds to then calculate the % cross reactivity.

good luck.




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