i need a nice protocol for celluler ELISA - (Oct/26/2007 )

I tried to make cellular ELISA but i got a very high back ground , may you advice me or help in a protocol you did by your self. Thank you

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Hello Manal,
I recently tried and failed as well with in-cell ELISA. Same problem as yours: terribly high background.
I've tried many things but none seemed to work: frequent washes, higher Tween concentration, low secondary Ab concentration,, but still the control (one NOT incubated with primary antibody) gave very high signal, almost as high as samples (ones incubated with primary Ab).
Can anyone help?

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Can you describe how you're performing the detection? With a little more info I might have some suggestions.

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you can use H2O2 in case u use HRP-conjugate. It inhibits intracellular peroxidases, it is very important if you don`t use harsh fixing conditions

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