I think your question may be a bit off. the object of using the Biotin
-avidin system is to be able to label a primary antibody on the cell
surface. You do not use a secondary antibody rather avidin conjugated
to a
fluorochrome.
The theoretical increase in fluorescence that is tauted for this
complex is based on the chemistry of biotin. the biotin molecule is
capable of binding several (I believe 7) avidin molecules. This is what
gives you the increased signal since now every biotin molecule on your
primary antibody is capable of binding 7 molecules of avidin ie 7
fluorochrome molecules. This is often more molecules than are attached
to
the secondary antibodies used for primary detection.
One should remember however, that more is not always better.
Fluorescent molecules may be quenched when in close proximity to other
fluorescent molecules and therefore the net result may be lower intensity
signals.
We have used biotinylated antibodies in three and four color analyses
since this gives us the option to use the second laser to detect the
avidin
binding using APC. in our hands, the biotin avidin antibody staining is
often at least 1 log brighter than the same antibody directly conjugated
to
the same fluorochrome.
There are clearly experts in this society who have a better
understanding of this area than I so you will want to assess all
answers to
this question.
Charles A. Kuszynski, Ph.D.
Assistant Professor/Director
University of Nebraska Medical Center
986495 Nebraska Medical Center
Cell Analysis Facility, WH 3024
Omaha, NE 68198-6495
402.559.6267 voice
402.559.4077 fax
ckuszyns@unmc.edu
This archive was generated by hypermail 2b29 : Sat Mar 10 2001 - 19:31:09 EST