biotin-coated plates in elisa - (Feb/24/2010 )

so a gal in my group is developing an ELISA and having a heck of a time.

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
2. block
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.

A

streptavidin is sticky. I have only heard of people biotinylating their abs...you are going a different route.

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.

we're looking for it in serum because patients will be treated and we need to monitor their serum levels. patients will be injected with Ab-SAV conjugate; afterwards we'll get blood samples which need to be assayed for the Ab-SAV concentration. at this point we're trying to develop a standard curve.

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.

Hi Aim…

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.

I like that idea...biotinylate BSA both are inexpensive.

hey, so we're getting much closer. we're using a biotinylated Ab to coat the plate first, and for whatever reason her results are improving. the 'noise' has gone down enough to get a respectable standard curve and she's fine-tuning now.

thanks!