Good morning, my name is David.
We have a protein structure and now we are looking for epitopes into the structure ( lineal and structural epitopes). When those theorical epitopes are achieved we want to test them experimentally. If there are lineal epitopes, we can do a ELISA with the incubation of epitope with human pacient serum. But Anyone know how test a structural epitope? Its possible to do a mutation into the structural epitope and test with the previous method? or the ELISA signal will be very low or masked with the rest epitopes?
Thank you a lot!
If you can isolate the whole protein in a folded state, you can test for structural epitopes by ELISA or other immunological methods that keep the protein in the native (folded) state.
As with everything else, it depends. If the epitope is immunodominant then yes, a single mutation may decrease binding significantly even for polyclonal antisera. Have ready the analytical setup for determining the effect of the mutation on folding kinetics/dG; you'll get quite some false positives.
In my opinion, you'll get less headaches by simply making hybridomas and then characterizing those individually. It's a lot of work but if you can afford it, the data is much more solid.
If your experimental objective requires accurate and high-resolution conformational epitopes, you can also consider using structural biology techniques for epitope mapping analysis. I think knowing specific structural information is more convincing than other biochemical experimental results. If you don’t have sufficient conditions to perform structural biology studies, you can consult Creative Biostructure, which is a professional supplier of structural biology technology as far as I know.
Hope it helps you.
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