I presume that the concept underlying the subtraction is that some
fraction of the binding of the monoclonal antibody is "non-specific",
i.e., binding to things other than the antigen of interest. Why not
eliminate this nonspecific component by pre-incubating the cell sample
with an excess of isotype-matched control, or some appropriate
equivalent, and then completing the staining with the antibody of
interest in the continued presence of the matched control? It should
be easy enough to determine to what extent this would reduce the MESF
values for cells expressing low amount of antigen.
Gib Otten
Chiron Corp.
Emeryville, CA
______________________________ Reply Separator _________________________________
Subject: MESF and correcting for isotype matched controls
Author: Calman Prussin <CPRUSSIN@niaid.nih.gov> at SMTP
Date: 1/6/00 11:42 AM
We are quantitating cell associated antigens using standardized beads and
then generating an MESF from the standard curve generated from the beads.
The question has come up whether or not to "correct" the MESF values
obtained from the specific antibody by subtracting the MESF values obtained
for the isotype matched controls.
The problem with correcting the values is that it has little effect on the
samples with high density of Ag, but has a large effect on those with low
expression. As such, if we run replicate samples on the low expressing
samples we find a larger amount of variation in the corrected MESFs. My
bias is not to use it, as it seems to introduce more "noise" into the
system. Your thoughts?
Thanks!
> _______________________
> Calman Prussin
> Laboratory of Allergic Diseases
> NIAID/ National Institutes of Health
>
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:02 EST