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I put out a note regarding this issue a couple of months ago and received no
responses. Hopefully, that was because the note never made it and not
because there is no one who has an opinion on this issue. We are trying to
compare the number of binding sites on native and stimulated neutrophils
compared to tranfected 3T3 cells. It is a human antigen, expressed on neuts,
and transfected into murine 3T3 cells. We are using a f(ab')2 fragment,
indirectly labelled. We are using the Quantum Simply Cellular beads from
FCSC. Our problems are multi-fold. 1. The background on 3T3 cells compared
to neuts is about a log greater. 2. Since in order to use the QSC beads you
have to put all 5 peaks on scale (at least the top 4), we have to put the neg
3T3 right about the end of the second decade, sometimes into the third. This
generates a background MESF of around 25,000 units. If the ABC Threshold is
267 units, does this mean that a sample that gives an MESF of 25,270 units is
significantly positive?
Dr. Schwartz says that these issues are not problems at all. But I am terribly
uncomfortable with the whole situation.
Here's the list of questions:
1. Has anyone else worked with Quantum Simply Cellular beads?
2. Does anyone have an opinion regarding comparing antibody binding capacity
of two such very different cell types, with or without a standard curve?
3. Does anyone have any suggestions as to alternative methods of quantifying
the number of sites. Iodination is not an option.
I greatly appreciate any help, suggestions, and/or advice.
Kris Weber
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