dual label assay
Becky Bonner (Becky.Bonner@CCLINK.NET.uokhsc.edu)
Tue, 08 Mar 94 16:19:33 CST
A week or so ago I had sent an email re a double label assay involving
an IgM and an IgG1 antibody. Thank you for your responses. I thought
you might be interested in our results so here they are:
dual label with IgG whole molecule assay secondary system binded
beautifully with IgM antibody when we switched detection systems (as
expected) i.e. IgM primary antibody + IgG biotinylated secondary +
neutralite Texas Red = great binding. Dual label system with
IgG1-primary monoclonal + biotinylated IgG secondary +
neutralite-Texas red followed by IgM primary monoclonary +mu chain IgM
secondary-Fluorx results were great. i.e. not all green cells were
red and not all red cells were green. in fact if we had'nt switched
the detection systems we wouldn't have known.
so we ordered gamma chain specific IgG from BRL and repeated the
assay. No binding of the IgM this time in the switched assay but the
regular labeling had a lot of nonspecific stuff and muddy looking ...
still not as good as we wanted.
so at one of the recommendations of the flow-cytometry users group
(Andreas Radbruch, Cologne University) we ordered biotinylated IgG1
from Southern Biotech to see if we could improve things.
and violah (don't know how to spell that word!) the assay is
beautiful. no observable cross reactivity with IgM (even though the
company says <1%) and a georgeous highly specific red signal from the
IgG monoclonal just as good as our standard IgG signal was. I guess
now I'm a sworn user of Southern Biotech reagents because it really
makes a difference.
thanks everyone for your help. the moral of the story surely must be
to be sure you all possible controls in a dual label assay. In this
case we did each separately, then together then swapped sencondary
systems in a single label assay. this was our original plan for
testing but we were sure surprised that the IgG whole molecule triple
label system looked so good with the tremendous amount of cross
reactivity we saw.
becky-bonner@uokhsc.edu
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