In article <bfnant$qf3$1 at reader1.panix.com>, iayork at panix.com (Ian A.
York) wrote:
> In article <3F1F1222.9000403 at NOSPAM.ucalgary.ca>,
> Bryan Heit <bjheit at NOSPAM.ucalgary.ca> wrote:
> >Mike Clark wrote:
> >
> >>Antibodies may or may not be "specific" depending upon how they
> >>are selected and how in practice you define specificity. Specificity is not
> >>
> >But how many times have you run a cell lysate blot with an antibody and
> >seen greater then 1 line? It's rare, and when it does occur it's
>> Many, many times.
Yes (esp. with polyclonals).
>> >usually human error or a closely related protein. In all of the blots
> >and elisa's I've done (that's a lot) I've never once encountered an
> >antibody with cross reactivity for a non-related protein, and that
> >includes some "dirty" polyclonal serums I derived myself.
>> Many of the polyclonals I've raised, and many of those we've got from
> various collaborators, cross-react. Rabbits see *lots* of antigens even
> in their constrained little lives in a lab cage, not just the ones you
> inject. Those may not be formally cross-reactive (i.e. the same clonal Ig
> may not be reacting with two antigens) but the result is the same.
(Exactly...I should have read this response from you first).
>> I've also seen a few monoclonals that cross-react with unrealted proteins,
> though not very many.
>> >Can you cite a single occurrence where antibodies show cross-specificity
> >with a protein only 50% identical, unless it is recognizing a shared
> >epitope? I did a quick check of pubmed and could not (not to say there
> >isn't, I didn't look too closely).
>> Most of the cross-reactive antibodies are simply thrown away, they're not
> worked up to the point of eliminating shared epitopes (an artificial
> constraint you've added to the original question). By the time they're
> published, you're generally only seeing the best (i.e. most specific)
> antibodies. Depending on the published literature for this question is
> not particularly useful.
Absolutely, because most people do not have a use for these and therefore
they are disposed of. In the cases where a broadly cross-reactive
monoclonal is DESIRED however, like I said before (a previous post?) it's
all a matter of what you choose to pressure the selection in the course of
screening. I happen to be developing hybridomas of this exact sort at the
moment...it makes screening rather tedious, but you get what you seek.
>> >Or you could look at it as a group of antibodies all recognizing the
> >same antigen. Idiotype antibodies are a special case, as it is hard to
> >identify what is the "antigen" and what is the "antibody". As I said
> >previously, I am unaware of any study showing cross reactivity between
> >proteins with little/no homology.
>> I've certainly seen cross-reactive monoclonals. Were they reacting with
> shared epitopes, with closely-related proteins, with unrelated stuff? I
> have no idea. I threw them away, because they were useless for my
> purpose. That's what happens to most of the cross-reactive antibodies.
>> Ian
best,
M.H.
