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> Dear Fellow Colleagues,
> We have a question regarding fluorescence intensity response at antibody
> concentrations beyond saturation.
> I have been performing antibody titrations with both directly conjugated
> and purified unconjugated antibodies followed by various secondary
> reagents and I am occasionally noticing a reduction in fluorescence
> intensity with increasing antibody concentration at levels beyond
> saturation. We have not been able to correlate this phenomenon with any
> variable, ie. fluorochrome, antibody isotype or cell type. The
> concentration range is from 0.05 to 50 ug/mL primary antibody, when a
> secondary is used its concentration is kept constant. Typically, we see
> a sigmoidal fluorescence intensity response with increasing antibody
> concentration. At high concentrations the intensity will level off and
> then begin to fall, sometimes precipitously. One feature we have noticed
> is that this generally occurs when the secondary antibody is a goat
> anti-mouse IgG-PE or FITC and not with SA-PE or SA-FITC.
> Antibody quenching could be a cause except that beyond saturation there
> should not be any additional binding taking place.
> I would appreciate any comments regarding this phenomenon. Many thanks in
> advance.
>
> Mark Rehse
> Rehsema@CellPro.CellPro.com
> (206) 489-8850
>
The phenomenon you describe is one that I have come across many times
using many different antibodies and different assay systems including
ELISA assays, radio-binding assayscas well as fluorescence assays.
I think that the explanation for this is associated with the fact that
monoclonal antibodies when used as bivalent IgG for example have the
ability to bind with high avidity bivalently and with lower affinity
monovalently. However in theory you can get twice the saturation levels
of monovalent antibody as for bivalent. In practice of course what you
get is a mixture of the two types of binding going on, and this will also
be dependent upon othr factors such as antigen density and steric
constraints due to antigen or antibody isotype (eg hinge differences).
At low antibody concentrations the bivalent high avidity binding will be
favoured. As you increase the concentration of input antibody you will
tend to force monovalent binding to be more favoured as the antigen
binding sites become more saturated more rapidly (ie imagine the
probability that an antibody binds first by one Fab and then what is the
chance of the second Fab encountering a free antigen). Now because we are
not working at equilibrium in our experiments, as soon as you start to
wash off unbound antibody you will get a situation where the higher
levels of monovalent antibody bound with lower affinity is actually lost
faster than the bivalent antibody bound with higher avidity. Hence you
see this "prozone" effect whereby higher input antibody results in less
bound at the end.
You mention that the secondary detection reagent also has an effect in
the system. I have also seen this before. I suspect that what is
happening is tha the secondary reagents may have a different capacity to
crosslink the primary antibody and hence increase their avidities. Thus
you may in fact be losing antibody from the surface all the while you are
incubating with second antibody. The lower the avidity of binding the
more rapid this will be. However if you crosslink two monovalently bound
antibodies you effectively make them behave more like one bivalent antibody.
I have done a lot of work on monovalent and bispecific monoclonal
antibodies and the observations seem to fit the above explanations. These
effects are more likely to be seen with lower affinity monoclonal
antibodies and antigens at high density.
Regards,
Mike Clark, mrc7@cam.ac.uk http://www.path.cam.ac.uk/mike_clark/
-- o/ \\ // || ,_ o Dr. M.R. Clark, Division of Immunology <\__,\\ // __o || / /\, Cambridge University, Dept. Pathology "> || _`\<,_ // \\ \> | Tennis Court Rd., Cambridge CB2 1QP ` || (_)/ (_) // \\ \_ Tel. 01223 333705 Fax. 01223 333875
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