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1) Affinity of the Antibody
2) Specificity of Antibody
3) Fluorochrome/Antibody Ratio
4) Machine Settings During Specific Run
5) Incubation Variables
Time of incubation with antibody
Temperature of incubation
Length of incubation
Number of cells in assay tube
Volume of antibody in assay tube
Concentration of antibody in assay tube
Wash Conditions
Post Wash treatment
Fixation of sample
Storage of sample
While this seems like a rather depressingly long list of things which can
make ones measurement of "quantity" of antigen vary, you must keep all of
them in mind because they can all effect your results, sometimes
drastically. The key to good measurements is setting up the proper controls
so that you are likely to be measuring the variable that you think you are
measuring. In many cases the experimentalist need not have an absolute (ie
molar or mg) measurement but just wants to get a relative measurement, say
the "amount of antigen under condition B is twice that under condition A".
If what one wants to do is to set up an assay in which the same cells are
being measured under different conditions but stained at the same time, and
one is assuming that the same antigens are on the cells (not addition of
others that may partially or strongly crossreact with the antibody) then it
is relatively easy to set up an assay to measure relative amounts of
antigen. There are, however some details that must be thought of such as
whether the size of the cells is drastically altered, which could in theory
decreaase peak fluorescence intensity with the same "amount" of antigen on
the cell. Having a "control" antigen which is known not to be modulated
would be helpful in this regard. In any case it can be done. Would strongly
recommend reference beads to minimize machine setting variables. The issue
of incubation variables is also very very important and makes comparison of
results obtained from different incubations more variable. With some
fluorescent antibodies and cells, nonspecific staining is a problem.
Edgar L. Milford, M.D.
Brigham and Women's Hospital
Tissue Typing Laboratory
75 Francis Street, Boston, MA, 02115
Tel: 617 - 732-5872
Fax: 617 - 566-6176
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