I am trying to develop a sandwich ELISA for my antigen. The commercially available antibody contains BSA in the formulation. Can I coat plate with this antibody or should I find one without BSA? Thank you.
Dear Wei
Depends on how much is the concentration of BSA vs antibody. In most cases both BSA and IgGs will bend. Try using a "high Binding" ELISA plate.
Dear Wei,
according to my experience it is always preferable to use "pure" antibody" for coating (without any other Protein which will block binding sites on the ELISA plate!!).
After determing the optimal coating conditions by testing different antibody concentrations, different buffers (e.g. PBS versus Carbonate buffer) and different pH, it will be necessary to block residual binding sites using a Protein-containing blocking buffer (with BSA, skim milk or FCS). For blocking you should always use a higher volume in the cavaties (e.g. 100µl for coating, 200 to 250 µl for blocking...)
All following steps might be done using this blocking buffer als a dilutent....
Kind regards
Wolf
It's advisable to comprehend principles of ELISA and its various types before venturing to experimentation.
This is a very important question as some companies deliberately only give antibodies with BSA and sell expensive ELISA kits. Did you try to coat with this antibody? What was the result? Thanks
Just don't use antibodies spiked with BSA for coating plates. The BSA will compete with the antibody to bind to the plastic and so you will not get a most out of your capture antibody. The best vendors will make BSA-free antibody available for your assay development. Make sure you buy known quantities of IgG, not volume!
It would be worth your while checking out the new free webinars on commercial research antibodies with priceless tips from leaders from both academic and industry. See https://www.antibodysociety.org/learningcenter/
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