Epitope mapping - (Jun/04/2007 )

We have generated a mutation in a domain of a gene of our interest that both mono and polyclonal antibodies are available. By surprise, we found out that this single mutation at the N-terminal, completely shut off our monoclonal, but not the poly. We think this domain should be part of the epitope to the monoclonal. I appreciate any comment and if please let us know how we can start validate the epitope by epitope mapping or else.

Thanks

hello,

as you said monoclonal Ab failed to detect your mutated protein, this indicates that the single mutation is important for your mAb clone binding.
where as the polyclonal Ab (unless you purify with peptide affinity column) can recognize multiple epitopes. that means your pAb recognizes multiple epitopes unlike mAb.

In this case you have to proceed with epitope mapping to confirm that your pAb can recognize different epitopes, then you will have the proper explanation.

good luck
sravan

hi,
as payeli said it's true that the mAb is binding at the region of mutation. it is generally true that pAb can recognize multiple epitopes. so i don't think there is any need to confirm the polyclonality by epitope mapping for pAb but ofcourse you should do so for mab. you also should make sure that the mutation is directly responsible for the non-binding of the mab i mean that there is no structural change by the mutation because of which the mab is unable to bind
all the best
leelaram

Hi,

You are right. This may simply be a structural change that has nothing to do with the mutation. We'll looking at it too. However, I am planning to make few mutations befor and after the original mutation to see if we can sequence the exact region. Do you think this is a right approach?
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Hi,
you can do that to map the epitope and also better do a western blot with the mutants so that the structural discrimination doesn't come into picture
all the best