2 antibodies from same host - same host ab's in sandwich (Oct/10/2005 )

I start doubting my sandwich ELISA setup, can the background be due to the fact that the capture and HRP conjugated AB's are from the same host?
will the background be lower when I use ab's from different hosts? and why is this?

Thanks!!
(sorry for the people who are reading this for the second time because I posted this question already under another (wrong) topic)

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Sounds like your problem is similar to mine. I was advised to try using a monoclonal ab for the capture step, then add my primary and secondary afterwards.
I can't actually say how well this works as I'm STILL trying to speak to my supervisor about it!

Rosie

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Mono-Poly combination is the preferred combination for sandwich ELISA

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Pity none of the protocals mention that!
or indeed the people selling antibodies!
ah well

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I'm not sure why that would matter so long as your HRP conjugated is specific to your protein of interest and not simply a secondary specific for Ig.
Try a host of different blocking agents, we had to try everything available to get one that gave reasonable background in our sandwich ELISA (Casein alone in this case as even Super block gave bad baskground)

good luck

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