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I agree with your concerns about specificity of staining. In staining
for intracellular cytokines, I've found that mAbs do not cleanly
saturate or demonstrate a plateau, as do with fresh cells. My
explanation of this is fixed permeabilized cells are sticky and at
higher Ab concentrations, non-specific staining becomes a major source
of binding. We have used 2 approaches to demonstrate specificity,
neither of which truly prove that the Ag being recognized is indeed that
which you are seeking. The first involves preincubating your mAb with an
molar excess of recombinant protein, I'm not sure if you can do it with
bcl-2. The second involves preincubation of your cells with "cold" or
unlabelled mAb prior to staining with FITC labelled mAb.
Ultimately, I've used this 2nd type of "cold" mAb block using 2
different mAbs that recognize separate epitopes on the IL-5 molecule to
demonstrate that the same cells are staining for 2 different IL-5
epitopes specifically. It is pretty hard to imagine a single molecule
that cross-reacts with 2 separate epitopes of your Ag of interest. See
JIM 188, pgs. 117-128, (1995).
So can you get a second good mAb to bcl-2 that recognizes a separate
epitope from the one you have?
>_______________________
>Calman Prussin
>Laboratory of Allergic Diseases
>NIAID/ National Institutes of Health
>
>----------
>From: david_hedley@pmh.toronto.on.ca
>Sent: Wednesday, September 18, 1996 15:16
>To: Cytometry Mailing List
>Subject: Quantification of bcl-2 by flow cytometry
>
>
>We are trying to quantify bcl-2 in human leukemic cells using a direct FITC
>conjugate from DAKO. Compared to the isotype control we get plenty of
>fluorescence, as has been reported by others. However:
>
>1) We do not achieve saturation using concentrations of up to 80ug/ml.
>
>2) We compared labelling of bcl-2 transfectants versus neo controls, and
>obtained only about 50% increase. Using the same antibody, western blots
>showed much greater differences.
>
>We have done a lot of work on this assay, examining different cell types,
>batches of antibody, and a wide range of fixation and permeabilization
>procedures. Depressingly, the antibody dilution curves stay the same.
>
>My working hypothesis is that the Dako antibody is cross-reacting with
>some other intracellular antigens; perhaps another member of the bcl-2
>gene family. However, I would be grateful for any ideas from people who
>have a reliable method up and running, or who may have any thoughts.
>
>David Hedley
>Princess Margaret Hospital/Ontario Cancer Institute
>Toronto
>
>
>
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