In article <001c01bf6d23$d27aa840$0400a8c0 at cweir>, Chris Weir
<URL:mailto:chrisweir at biotechfrontiers.com> wrote:
> Hi All,
> I've been working on developing monoclonals to carbohydrate antigens.
> One in partic ular has given some interesting results. Initially all we
> could produce was IgMs to th is antigen (As is the case with alot of
> these antigens). After developing a method of presenting this antigen to
> mice we produced a strong IgG response and after a fusion t he first IgG1
> to this antigen.
> This IgG1 will outcompete any of the IgMs/IgG3s we have for binding to
> the epitope. T he IgG1 however has an extremely high-offrate compared to
> the IgMs which once attached bind strongly. - In my experience (and in
> general) IgG1s have a hi gher affinity than that of IgMs - but not in
> this one.
> I have one theory - The actual protein which has the Carbohydrate
> epitope is around 3 00 Kda and has heaps of these carbohydrate epitopes
> on it. I've talk to some protein e xperts who have said it is likely that
> the sugar epitopes are probably slightly different across the whole
> structure. Which has led me to believe that maybe why the IgMs have a
> lower offrate is that they can get more arms onto the varying suga r
> epitopes - hence bind better thus have a higher affinity because the can
> bind to more than one site.
> If anyones got any other Ideas - I'd be glad to hear them.
>> Regards
>> Chris Weir
> BTF
My immediate reaction to the above is that this is what you would expect
where the valancy of binding is greater than two for an IgM. The IgM will
have a higher avidity for antigen and hence a slower off rate.
To put it another way. Suppose in an immune response you had the identical
V-regions in a pentameric IgM and an IgG. The on rate for binding for
the first binding site would largely be dependent on the rate of diffusion
of the molecules. Therafter the second and third and fourth binding sites
are more likely to be influenced by the valency and spacing of the
antigenic epitopes.
Once several binding sites are engaged the off-rate for the whole molecule
is determined by the product of the off-rates for the individual binding
sites.
Thus by definition most IgG antibodies which bind to multimeric antigen as
readily as an IgM are of higher affinity. Another good experiment is to
take an IgM and treat with mild reducing agent. The subunits fall apart and
it has a similar valency to IgG. Many IgM antibodies when treated this way
have too low an affinity to detect binding to their antigen.
Regards,
Mike
'The immunoglobulin structure and function website'
<URL:http://www.path.cam.ac.uk/~mrc7/>
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