pre-immune serum came out positive

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Thu Feb 15 10:57:42 EST 2001


Regardless of immunity that may arize after immunization, I think we'd all
agree that it is prudent to look at preimmune serum prior to immunizaion.
Afterall, in the original post, the concern was that pre-immune seemed to
be reactive to as many proteins as immune.  Checking serum before
immunization may not solve all the problems, but it will avoid starting out
with one!

Mike


>Henk Veldman <H.Veldman-2 at neuro.azu.nl> wrote:
>:"Michael L. Sullivan" schreef:
>:
>:> Well, your rabbit was probably exposed to something that immunized it
>against
>: proteins in your extract.
>:>
>:> What were you using the antibody against, some sort of crude extract
>and what
>: was the extract made from? I know that plants and yeast often have proteins
>: that cross react with antibodies in pre-immune serum. For plants, it is
>maybe
>: related to diet, which contains plant materials, and for yeast, I hear it's
>: because rabbits often have yeast infections. I don't know how prevalent
>cross
>: reaction is with other types of extracts.
>:>
>:> It is a good idea to test preimmune serum from rabbits *before*
>immunization
>: with your antigen. When. . .   <......>
>:
>:Even when the preimmune serum is clean, the immunization procedure, usually
>: with some kind of adjuvant, can induce immunity against unwanted  antigens.
>: Most notably quite high titers might be raised against keratins from human
>: skin that are around everywhere and are also difficult to avoid
>completely in
>: protein preparations for SDS-PAGE / western blotting.
>:Such antisera can be perfectly OK for some applications such as
>: immunohistochemistry, otherwise affinity purification may be necessary.
>
>Absolutely correct. Anyone who ever tested preimmune serum vs
>immune against irrelevant antigen vs immune against the antigen
>assayed can confirm this. Adjuvants boost immune response
>against _all_ antigens.
>
>That is exactly why using preimmune serum, even when from
>the same animal, is in fact not a very thorough control. I've seen
>a number of screw ups where claimed "specific something"
>persisted upon serum/IgG immunodepletion on an antigen
>column. Of course, doing such right controls is difficult so
>almost nobody bothers with them and usually a non-immune
>serum from a different animal is considered to be an accurate
>control.
>
>        - Dima


Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax


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