Biology Reference
In-Depth Information
as the Alexa series from Molecular Probes may have increased photostability
and less self-quenching, it is still not possible to ensure a uniform conjugation
ratio with these small molecular dyes.
Direct Versus Indirect Conversion
Calibration using the QSC system allows direct conversion of MFI into ABS
per cell whereas the QB system converts MFI into the number of PE mole-
cules bound per cell, again indicating the importance of using a ¯uorophore/
antibody conjugate ratio close to 1. Puri®cation of 1:1 conjugates is usually
done by HPLC puri®cation of PE-conjugated antibodies, using fractions that
give a 565 l/280 l ratio of 3.4 G 0.2. Most commercially available PE con-
jugates may contain mixtures of multimers (2:1 to 1:2) and 1:1 conjugates.
The ABS value obtained for a speci®c antigen also depends on the valency of
antibody-antigen interaction, which can only be determined experimentally.
Another presupposition in using the QSC system is that the stoichiometry of
binding at high antibody densities is the same on the beads as on the cell.
Reproducibility
Quantitative FACS analysis allows data to be compiled and compared from
multiple experiments without having to rely on subjective interpretations of
slight shifts in MFI. In our experience, using the Kolmogorov-Smimov ( K-S)
statistics to determine if slight shifts in histogram pro®les are signi®cant can be
misleading; we have done experiments to show that two isotype controls when
overlaid can sometimes give p values of <0.05 when using the K-S statistics
pro®le on the CellQuest
TM
(Becton-Dickinson, San Jose, CA) program. How-
ever, QFACS relies on a strict set of predetermined parameters, which, when
satis®ed, allows one to place a degree of con®dence in the numbers generated.
When using QFACS, the above-mentioned isotype controls gave ABS values
within the standard error stipulated by the regression curve generated and thus
were not considered as signi®cantly di¨erent.
Quantitation of cell surface antigens is only meaningful when results can be
compared between laboratories. Therefore, for meaningful comparisons to be
made, ABS values can only be compared when using the same monoclonal to
the same cell surface antigen. Any extrapolation to the number of actual anti-
genic sites requires experimental determination of the valency of antibody
binding.
QFACS ANALYSIS FOR CORECEPTOR EXPRESSION
For quantitative determination of coreceptor expression levels, it is crucial that
the most appropriate antibody be used. There is increasing evidence that CCR5
