Custom polyclonal peptide antibody services? - (Jul/09/2008 )
Hi all --
Has anyone used one of the many services which will raise antisera against a synthesized peptide, selected as an antigen on the basis of physiochemical properties of the protein of interest?
See, for example, services offered by:
The ability to select an antigenic peptide from an amino acid sequence and raise antisera to a synthesized version of this peptide is attractive, as it would save us all the up-front labor and associated costs of PCR amplifying and cloning a gene into a his-tag vector, over expression and purification of the fusion protein, shipping it off for injection, etc. -- our current approach for obtaining polyclonal antisera.
But (there’s always a but...), we’re a bit concerned about the peptide antisera not actually recognizing the native protein, the accuracy of epitope prediction, and the possibility that the sera will be of only limited usefulness (e.g. it will work fine in westerns, but fail in immunoprecipitation, ELISA, or other applications).
What experiences do BioForum users have with such services? Are there pitfalls to avoid? Good companies, bad companies?
Thanks for sharing any experiences...
I haven't used any of them for that particular service, but... If I had to pick...
I definitely trust both Invitrogen and Sigma...I use many of their products, have never had a complaint, AND their technical help desk are awesome... That really is very important...
I don't know about the other two...I trust them both quite a bit but if I HAD to pick one to go with today, my instincts (regarding antibodies) would be to go with invitrogen...
by the way,
I'd be very interested to see how it works out for you... Would you mind letting me know?
You may have progressed by now but here's my thoughts for you. I should first declare an interest as I work for Cambridge Research Biochemicals . Anyway selecting a peptide as an antigen can be a highly sucessful approach but you are right to be slightly cautious. A lot of peptides selected as antigens are just picked just using some fairly standard algorythmns none of which are 100% effective.
There are important things to consider when selecting the peptide over and above whether it looks "antigenic" by the standard methods.
Is the peptide good from a peptide synthesis angle, tougher sequences will lead to more impurities and potentially higher backgrounds.
Are PTM sites in the selected peptide, it is no good raising an antibody to a peptide when the native protein is phosphorylated, glycosylated etc in your samples.
Is the peptide chemically stable or will it degrade during the immunisation process leading to the animal being immunised with a different antigen. For example Met containing sequences or this prone to aspartimide formation.
Is the protein transmembrane and if so is the antigen selected suitable for your application, intracellular sequences can look great in antigen selection proteins but be useless in your application. As you mentioned they can work in one application and not another.
Are there other isoforms and if so will the antigen recognise these.
Just a few thoughts for you there, feel free to get in touch if you want more info "off-line"
Thanks so much for your thoughtful comments -- this is exactly the type of information I was looking for. When I saw your company's name, I was hoping for Cambridge, Massachusetts, USA (thus right down the street from me), but alas, it was not to be...
We've had a company give us a quote and along with the quote they provided candidate peptides (14-mers) for each of our five proteins, with their recommended peptide highlighted. I've now run our proteins amino acid sequences through several different prediction servers (such as BepiPred, (which uses a hidden Markov model and a propensity scale), BCEPred (which seems to be down right now, but makes predictions using a combination of hydrophilicity, flexibility, accessibility, turns, antigenic propensity, exposed residues, and polarity scales), and BCPred, (using all three methods they allow there: the AAP method [Chen et al., 2007], the BCPred method [EL-Manzalawy et al., 2008], and the FBCPred [EL-Manzalawy et al., 2008]). People really need to come up with some more imaginative names for their linear B-cell epitope prediction servers, huh?
I've mapped all this data to our amino acids sequences, and overlaid this with things we know about the particular regions of the sequences (glycosylation sites, for example). I've then plotted a histogram with residues on the X-axis versus the number of methods that selected a particular residue as being a candidate for an epitope.
I know it's not strictly a "majority rules" selection, and I am, of course, brand new at this, so I'm just making it up as I go along -- but I was interested in seeing how the company-recommended peptides fared as far as publicly available (and perhaps more current) methods were concerned. There is considerable overlap in the regions selected, but there are also regions that look good by one method and are completely ignored by others.
You raise an excellent point about "rubber meets the road" issues like the peptide's ease of synthesis and chemical stability -- I have virtually no information on these no doubt critical considerations. Can you point me at some further information that might help me judge whether the peptides I'm thinking about having synthesized will actually perform in a reasonable manner from a technical point of view?
I've also read that selecting two peptides from a particular protein's peptide candidate list, having them both synthesized and then injected as a mixed immunogen can maximize the chances of producing a more useful sera. Any thoughts on this approach?
Sorry for the delayed reply (see my pm for more info) but here are some quick thoughts. I'll try and flesh this out later but a bit busy today.
Some general things to avoid in antigen peptides:
Internal Methionines - these could oxidise so the animal is presented with an incorrect immunogen.
Areas with multiple Asn, Gln, His and Arg can reduce purity.
Internal Cysteines - If not used for conjugation these are 1) probably part of a di-sulphide and hence the structure could be way off and 2) could dimerise with other peptide ruining the immunogen
Multiple Asp or Asn residues next to each other can lead to aspartimide formation again modifying the antigen.
N-terminal Gln should be avoided where the antigen is conjugated at the C-terminal as this forms pyroGlu, acetyaltion of the N-terminal will avoid this
Peptides with long hydrophobic areas can be difficult to purify.
Lot's of other factors as well but essentially antigens shoudl be assesed by a peptide chemist as well as a biologist if at all possible.
The multiple antigen approach I have severe negative thoughts about. Whilst technically this is potentially increasing the chances of protein recognition it is also potentially doubling the impurity profile and hence the chance of cross reactivity to something else in your samples. If this occurs deconvulation is tricky. It is far better to use 1 antigen per pair of animals. This appears popular at the moment for budget reasons but is not a good idea.