Re: Ab titrations - effect of Ag density in a single cell

Uriel: you are correct that in theory the antigen density on the cell 
surface could affect the titration.  In fact, far more than that is 
relevant: it is quite possible that the conformation on one cell is 
different than on another, leading to different titration values. 
This is seen with antibodies to CD8a, which have a different avidity 
when complexes as homodimers (e.g., on NK cells) than as a/b 
heterodimers (e.g., on most CD8 T cells).  In fact, what people refer 
to as "dim CD8" T cells usually express just as much CD8a as "bright 
CD8" T cells--they are just not using the antibody in saturation.

Hence, when you titrate an antibody, you determine the appropriate 
concentration for your particular cell type, your particular 
conditions (temperature, fixative, buffer, etc.), etc.	Do not assume 
that if you change any of these parameters that the titration will 
remain the same.

Nonetheless, practically speaking, changing nothing other than the 
antigen density several fold (even 10 fold) on a cell will not affect 
the titre dramatically.

Yet another reason to remember to "Titrate, titrate, titrate".

mr

>Dear friends,
>
>I have a question regarding antibody titrations that I wanted to ask 
>"the collective". I've read the list's archives but still it is not 
>completely clear.
>Assuming from now on that temperature and incubation time are left 
>constant; it is clear that the most important factor is the 
>concentration of the Ab and the cell number is irrelevant most of 
>the time (except when the number is many times more than titrated or 
>the Ab is so avid that at saturating conditions it is not in huge 
>excess). But what about antigen abundance? Quantitatively, one cell 
>expressing 2X antigen is like two cells expressing 1X antigen, so 
>the "cell number is almost irrelevant" should apply.
>But then one cell with 2X antigen would have 2X the antigen 
>concentration per surface area, and given that Abs are bivalent 
>(except IgM of course but lets leave that aside for now) that would 
>change things regarding the actual observed avidity of the antigen, 
>which is what we "use" (as opposed to the affinity of a single Fab 
>which would stay the same). It could be that now the Abs can bind 
>"better" the cells since every bound Ab has more of a chance of 
>finding a second available antigen, or it could be that the 
>"monovalent binding vs bivalent binding" competition ensures. 
>The interaction between these two options, which one happens when is 
>unclear to me. Regarding the antigen concentration, it would seem a 
>possibility that, in analogy to enzymatic reactions where binding of 
>the substrate increases its local concentration for subsequent steps 
>done by coupled enzymes or active sites, once an Ab binds 
>monovalently, the binding of the 2nd idiotype would be affected by 
>the local concentration or availability of the antigen. Then 2X 
>density on one cell would be different that 1X concentration in two 
>cells, even when overall the antigen concentration is the same (at 
>least when bivalent binding is involved). All these questions are 
>related to the fact that the archive discussions and books that i 
>have found use a monovalent binding model, and i wonder if it 
>applies in the case of "local" (cell surface) antigenic density 
>increase in contrast to soluble Ag kinetics.
>
>All this of course has implications when titrating and using Abs: do 
>I have to titrate using the cells that express the Ag at max? or 1/2 
>max is enough? I.e. immature DCs vs activated DCs when titrating DR 
>or CD86. Furthermore, would it be OK to use monocytes to titrate and 
>use the results for DCs? Or, since monocytes express considerably 
>less DR and CD86 than DCs, say 1/3, would it be OK to titrate 
>300,000 monocytes and assume that the amount found is good for 
>100,000 DCs? (these are just examples for the sake of the argument).
>
>Insights and clarifications are most welcome!
>
>best regards,
>
>Uriel
>
>PS: I will post a summary of the responses.
>
>Uriel Trahtemberg
>MD/PhD student
>The Laboratory for Cellular and Molecular Immunology
>The Hebrew University - Hadassah Medical Organization
>Jerusalem - ISRAEL
>
>
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