In article <3ddhgc$3q7 at newsbf02.news.aol.com>, sbehar at aol.com (SBehar) writes:
> You will definitely get anti-BSA antibodies, and there is no easy way to
> avoid this in the production of polyclonal antisera (although substractive
> immunization is a possibility but better suited for mice and the
> production of monoclonal antibodies). However, one useful strategy is to
> immunize with the peptide conjugated to a carrier such as KLH, and then
> assay the antiserum using the peptide to another carrier such as BSA.
> This way, the antibodies raised against the carrier will not be detected
> in the assay (for example, in an ELISA).
>
Careful! If you use the strategy outlined above you will have to choose
a different coupling for the conjugate used for assay compared to the conjugate
used for immunisation. We have found substantial cross-reactivity that is
entirely due to the chemical linker (be it carbodiimide, glutaraldehyde or
MBS) and under certain circumstances we have found cross-reactivity between
the linkers (!). We found that the best way to get antisera suitable for
assay was to either back-absorb with a conjugate of the immunising carrier
coupled to an unrelated antigen (or a fragment of the immunogen) or to
affinity purify the required antibodies. The latter is not as straightforward
as it seems as it only seems possible to elute a subset of positive antibody
species from the affinity matrix - and some of the best are almost unelutable.
Make sure that you have all the necessary controls,
Bernard
(and all the best for 1995)
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
