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Your point about discrepant F/P ratios between directly conjugated control
and test antibodies adding another unwanted variable to further confound
the use of isotype controls is well taken. (Controlling an indirect assay
for NSB is even tougher, in my opinion, since you can't assume conjugate
binding to the primary control and test antibodies is identical.) Among the
assumptions we make when we use them is that the manufacturer is using the
same conjugation process on two antibodies of the same isotype with the
same fluorochrome so we hope that F/P ratios are similar. Just so, we also
hope/assume that the control antibody and the test antibody have the same
NSB to the test cell; most of us realize that to be mostly wishful
thinking.
After all this nitpicking, the fact remains that with all their flaws,
isotype control antibodies are still the best tool we have for evaluating
NSB. Certainly, many flow-ers expect too much of them. I'm ready to hear
that there is a better alternative!
Cheers,
Karen Henell
Histocompatibility Technologist
Laboratory of Immunogenetics and Transplantation
Oregon Health Sciences University
Portland, OR 97201 USA
henellk@ohsu.edu
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