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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
>
>
Dear Mark,
we have seen the same effect using GAM-FITC. Beyond the critical mAb
concentration the fluorescence intensity decreases using the same GAM-FITC
concentration.
My explanation is: In the lower mAb concentration (non-saturating) the
majority of Ig is bound using both binding sites to the cell surface Ag
while beyond the saturating concentration there is mostly monovalent
interaction between Ig and Ag. Therefore GAM-FITC (where some molecules are
directed against epitopes close to the binding site of mAb) could compete
with mAb-Ag interaction (which is more successful in the case of monovalent
mAb-Ag than bivalent mAb-Ag interactions) and you loose those mAbs from the
Ag during subsequent washing after GAM-FITC labeling. If this is true,
SA-FITC or directly labeled mAb should show classical titration curve
instead of sigmoidal one.
*******************************************
Karel Drbal
e-mail: drbal@biomed.cas.cz
Institute of Molecular Genetics
Academy of Sciences of the Czech Republic
Videnska 1083
142 20 Praha 4
Czech Republic, Europe
voice: +42-2-4752589
fax: +42-2-4727979
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