>We are quantitating cell associated antigens using standardized beads and >then generating an MESF from the standard curve generated from the beads. >The question has come up whether or not to "correct" the MESF values >obtained from the specific antibody by subtracting the MESF values obtained >for the isotype matched controls. > >The problem with correcting the values is that it has little effect on the >samples with high density of Ag, but has a large effect on those with low >expression. As such, if we run replicate samples on the low expressing >samples we find a larger amount of variation in the corrected MESFs. My >bias is not to use it, as it seems to introduce more "noise" into the >system. Your thoughts? Calman, I, personally, agree entirely with your logic. We wrestled with the same issue when we were quantifying TCR's on stimulated T cells (which has now fallen out of popularity). I always argued against normalizing against isotype quantitative values (for the reason you stated), and I used to use a silly analogy in the attempt to make this logic inescapable: If you and I were sitting in the stands at Veterans Stadium at night when the lights were off and you had your flashlight on, then I accidently turned mine on, the increase in the intensity of light would be two-fold. In this case, you and I are the background, and my turning on my flashlight represents a (two-fold) fluctuation in background due to noise. Now, if you again had only your flashlight on and we were joined by 998 fans, each with their flashlights on, and then I turned my flashlight on, does the intensity of light which fills the stadium go up two-fold? Of course not. It goes up by 1/1000th of the intensity before I turned my light on. But the former is what one would be implying by normalizing samples with high fluoresence/MESF to the fluoresence/MESF of background, and thus one introduces the potential for tremendous artifact. Now, as you imply, this analogy/argument begins to break down when the antigen density decreases (say to 3- to 5-fold above background. My opinion is that is that if you can show that the variance in the MESF of the specific stain from sample-to-sample is significantly less than the variance of the isotype control values, then it would be most appropriate to NOT normalize against isotype. These rationalizations make me feel better, I hope they help you also. AW Andrew D. Wells, Ph.D. University of Pennsylvania Department of Medicine 728 Clinical Research Building 415 Curie Boulevard Philadelphia, PA 19104 (215) 573-1840 (office) (215) 898-1951 (lab) (215) 573-2880 (FAX) adwells@mail.med.upenn.edu
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