peptides for immunization and ELISA
Posted 03 February 2011 - 09:13 AM
Sometimes we do peptide-carrier immunizations in our lab. For this purpose, we purchase the needed peptide with a cysteine in one end for carrier conjugation (usually to KLH or OVA). Needless to say, that cysteine does not belong to the proteinīs sequence. Later on, to assess serum titers, we do ELISAs using the same peptide. However, in my understanding, we should use the peptide WITHOUT the extra cysteine since otherwise we wonīt be able to distinguish antibodies raised against a sequence containing that cysteine from the antibodies of real interest. I explained this reasoning to my colleagues but they argued that buying two peptides is too expensive and that -anyway- almost no antibody will bind the cysteine since that amino acid is coupled to the carrier. On the contrary, I think the cysteine residue is quite hydrophilic and so it will be very immunigenic. I think it would help a lot to this discussion if I could show some published data supporting any of this views. Any papers you know, people?
Posted 03 February 2011 - 11:38 PM
Posted 04 February 2011 - 02:46 AM
In our experience, it is very important to design the peptide carefully in order to get successful antibodies. If you choose any structurally compromised peptide of the protein then I would expect it doesnīt work because the free peptide wonīt get the same structure. However, if you have crystallography data of your protein and you can identify the sequences belonging to loops or unordered fragments, then the chances of getting a good antibody highly increases. At least, that is the experience of our lab (I am new here). Thank you for the paper, I will read it.
Posted 04 February 2011 - 03:46 AM