biotin-coated plates in elisa
Posted 24 February 2010 - 02:49 PM
we want to look for a SAV-conjugated mouse antibody in patient serum. I think we should be able to make this work by:
1. coat plate with biotin
3. add serum (varying conc. of Ab spiked into negative serum)
4. add anti-mouse Ab + HRP
5. add color-reagent and detect
we're getting a TON of noise. we've tried every control I can think of - blocked with NFDM or BSA, varied concentration of Ab in serum as well as the detection Ab.
in order to achieve any kind of standard curve above the noise, we have to add a ridiculous amount of detection antibody (we can get a narrow curve at 1:100)
it seems there are a couple of possibilities. we've considered switching isotypes of our detection Ab, and also adding an extra step for signal amplification.
what else? I have the feeling I'm missing something obvious here.
Posted 25 February 2010 - 11:07 AM
Why detection in serum...you now also have to worry about heterophile abs, matrix effects etc.
Could you use a common anti mouse ab on plate; react with sample in buffer (why sera?), and react with biotin alk phos? This would be a different approach. you would be able to test your approach by using mouse Mab as sample and detection with goat/rabbit anti mouse alk phos.
Posted 25 February 2010 - 01:17 PM
we had thought of switching it up so the plates are coated with anti-mouse Ab and detection is done with biotinylated reagent, but we'd still be using the same reagents; if there's a problem with non-specific binding we won't be getting rid of it. the only rationale for performing the elisa in that order would be to increase signal amplification.
Posted 26 February 2010 - 07:03 AM
This is just a thought…instead of coating with biotin (small molecule) how about biotinylation an antibody (say rabbit, goat etc) or another carrier protein and use hat for adsorption in your ELISA.
Posted 02 March 2010 - 08:30 AM
Posted 10 March 2010 - 10:56 AM