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We've recently done a series of replicate titrations and are shocked
to see that the concentration giving 50% of maximum fluorescence
intensity (Chalf) varies by as much as 2.5-fold between replicate
dilution series, whether done on the same day or different days, and
even when the starting dilution (typically made from 25 microliters of
neat antibody) is in common between two parallel dilution series.
We typically use 3- or 4-fold dilutions in the series, transferring 25
microliters per step. We obtain Chalf by fitting the resulting
fluorescence intensities to 1-site Michaelis Menton kinetics, namely I
= C(Imax)/(Chalf + C), where C is the concentration of Ab, and Imax is
the intensity at saturation.
Reproducibility of intensities for standard beads from one day to
another is within a few percent (with FL1 PMV at the same voltage, log
amplification), and reproducibility of median channel values for
replicate readings of the same sample on the same day is within a few
channels. Signal to noise (Imax/2nd Ab only or autofluorescence) is
>=30. The standard deviation of weighed replicate deliveries from the
25 microliter pipeting device is about 3% of the mean.
Can anyone routinely do better than this for Chalf determinations? If
so, what do you recommend? (Just better dilution technique?) Or is
this the best we can expect?
--
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Eric Martz emartz@titan.ucc.umass.edu Professor of Immunology
Voice 413-545-2325, FAX 413-545-1578
Morrill IVN Rm 203, Dept Microbiol, Univ Mass, Amherst MA 01003 USA
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