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peptides for immunization and ELISA


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3 replies to this topic

#1 Chelo

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Posted 03 February 2011 - 09:13 AM

Hi!

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?

#2 BioMiha

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Posted 03 February 2011 - 11:38 PM

We have done a lot of peptide immunizations and honestly I would not do that again. I am attaching a paper from a very renowned professor in this field who has a lot of comments about peptide immunizations. In my opinion there is a very low chance of obtaining an immune response that will cross-react with the native protein from which the peptide was taken. I would very much advise against using peptides for immunizations. I have seen people with a lot of antibodies to the peptide, that just don't bind to the whole protein. Concerning the cys residue, I think it is not as important. The majority of the immune response is usually directed to the free (non-coupled) end of the peptide.

#3 Chelo

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Posted 04 February 2011 - 02:46 AM

We have done a lot of peptide immunizations and honestly I would not do that again. I am attaching a paper from a very renowned professor in this field who has a lot of comments about peptide immunizations. In my opinion there is a very low chance of obtaining an immune response that will cross-react with the native protein from which the peptide was taken. I would very much advise against using peptides for immunizations. I have seen people with a lot of antibodies to the peptide, that just don't bind to the whole protein. Concerning the cys residue, I think it is not as important. The majority of the immune response is usually directed to the free (non-coupled) end of the peptide.

Dear colleague,

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.

#4 BioMiha

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Posted 04 February 2011 - 03:46 AM

In my experience even if you know the peptide is part of a loop, there is absolutely no guarantee that the Abs will bind to the native protein. Most of the commercial suppliers of anti-peptide sera have a very important clause in their terms and conditions and that is that there is no liability for them if the serum does not react with the protein. I have heard of very frustrated researchers trying to force the companies to redo the immunizations and provide sera that will react with the protein but no go. In my experience it is not worth the time and effort. I would suggest immunizations with the whole protein and then select for the best Ab response.




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