Re: Flow cytometry vs. Immunoblotting and Enhanced Chemiluminesence
James W. Jacobberger (jwj@po.cwru.edu)
Thu, 15 Feb 1996 11:46:37 -0500
Dr. Salkind,
In work that we are doing, we have shown that the coorelation between
chem1iluminescence and flow cytometric immunofluorescence assays for SV40
large T antigen over a 50 fold range is linear, has a very high r-square,
and is reproducible (two labs). Chemiluminescence appears to be more
sensitive. I think the reasons for this are: 1) "non-specific" staining is
subtracted out by eliminating bands that are not the correct molecular
weight or mulitples thereof, 2) there is no scattered laser light leaking
through, 3) there is no autofluorescence, 4) there is no background from
overlapping emmission from other fluorochromes, and finally, (5) because
there is no background, the signal can be enhanced by concentration of the
sample and by accumulation of the signal over time.
>What are the relative attributes of detecting antigens (specifically those
>that are intracellular) by flow cytometry vs. ECL? In our system, we've
>found immunoblotting and ECL to be much more sensitive. Does anyone know
>the limits of antigen detection of flow vs. ECL?
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>Thanks in advance for your comments and ideas.
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