ELISA and BSA - Non-ELISA grade BSA (Jun/28/2006 )

I've been using 1% BSA as a blocking agent but have found that when plates are not blocked at all, the EU values are lower. We're testing human serum and I have just found out that the BSA that we've been using is not ELISA grade. How does this affect the EU data? Help?

I suggest that you use BSA from Sigma

do you mean the BSA produced by Sigma does better than any other corporations?

We use Sigma BSA. As far as I know it's not specifically ELISA grade but it's purified Fraction V.

We use it at 200µl 5% BSA/PBS to block for 1hr and then 1% BSA/PBS to dilute samples and standards (if they need diluting) and diluting the detection abs and streptavidin-HRP.

All the best,
Ceri

HI,
i m bit confused about ur method, if u donot do blcoking, then u must get really higher back ground, by which u cant even detect the difference between controls (primary antibody binds to every where in the well n ultimately will bind to sec ab to give high OD)

please could u clarify this doubt for me, and let me know how exactly u calculate ELU values?

i used to use fraction V bsa from roche. unless u have lot of impurities in the bsa ur using for ELISA, u shud get similer results.

u can do a simple experiment, use different percentages of BSA for blocking and check the EU values? if u can see dose dependent difference in ur EU value, then u can be sure that bsa is the culprit.

hope it made sense to u

gud luk
payeli




i donot think we can differentiate that

Hi everyone

i am also having a lot of problems with my Elisa assay, i have been trying to develop an ELISA, and nothing has been working yet, ie. trying to get an standard curve to work etc. basically nothing has been reproducible.

so after having talked with my AB company, they have brought up the idea of using an Elisa grade BSA,
i have been using BSA from Sigma and then making appropriate % BSA for blocking and for diluting my samples.

does anyone with more experience with Elisas know if the sigma BSA which is Fraction V is ok?

anyhelp would be appreciated

thanks alot

ali

Hi Ali,
We use purified fraction V of BSA ("B" for Bovine) from Sigma as a blocking reagent @ 3-5% BSA in PBS (200µl/well) and dilute the samples (if needed), standards and detection abs in 1% BSA in PBS. It works fine. R&D or BD ELISA kit protocols suggest FCS as a blocking reagent which I guess most labs will have but will work out more expensive.

All the best,
Ceri

another blocking reagent you cou could try is casein or sodium caseinate, whatever you want to call it, should solve some cross-reactivity problems anyhows