Antibody Saturation

Mike Clark (Path) mrc7 at
Wed Jul 12 04:22:58 EST 1995

On Tue, 11 Jul 1995 REHSEMA at wrote:

> 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 at
> (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.


Mike Clark, mrc7 at
  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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