Actually, both PE and APC will form larger-order complexes--because the Streptavidin conjugates of the PBP are often larger-order complexes (usually, about 3 or 4 SA/PE or SA/PBP per covalent complex). The reasons for this are many, including that it is easier to make than trying to isolate the 1:1 complexes. However, according to one manufacturer that I spoke with, their anecdotal evidence is that people generally "prefer" the preparations of SA-PE (and SA-APC) that contain these higher-order complexes compared to the 1:1 preparations--primarily because they are brighter than the 1:1 complexes. We have observed significant differences in brightness between tetramers made using SA-PE from different manufacurers--presumably because of the different content of higher-order complexes. As an aside, the presence of these higher-order complexes means that comparison of relative brightnesses of any given tetramer reagent on different samples does not imply that the T cells have different affinities (or, more properly, avidities) for the MHC-peptide complex. Rather, the only way to measure relative avidity is by doing a titration of the reagent and reporting the concentration that gives half-maximal fluorescence--a task that is extremely arduous considering the rarity of these cells. mr At 11:55 AM +0200 8/9/01, Voorn, J. wrote: >Dear Paul, > >Perhaps I can not explain the phenomenon you see with the APC conjugates but >at least I can confirm the same findings with some of our tetramers. We have >seen that APC sometimes functions better then PE with certain T cell >populations. We think that it might be due to the difference in size of the >final constructs, on the other hand it has been suggested that APC form >larger multimers then 4 (although without evidence). >I am curious to the experience of other MHC-tetramer users, more information >on our tetramer facility can be found on our web site www.clb.nl ><http://www.clb.nl> and go to "reagents". > >Best regards, > >John > >John Voorn CLB Reagents > > > >Productmanager Plesmanlaan 125 >j_voorn@clb.nl <mailto:j_voorn@clb.nl> 1066CX >AMSTERDAM > the Netherlands >tel +31-(0)20 5123246 >fax +31-(0)20 5123570 web site www.clb.nl <www.clb.nl> > > > > -----Original Message----- > From: PAUL HALLBERG [SMTP:Paul.Hallberg@mail.tju.edu] > Sent: Tuesday, August 07, 2001 5:57 PM > To: Cytometry Mailing List > Subject: MHC-Tetramer Assay > > > Hi FLOWers, > > Can anyone explain why APC-SA gives a better S/N ratio than PE-SA >for some > MHC Tetramer Assays? In addition, does anyone know an alternative > fluorochrome conjugated to strepavidin using 488nm laser excitation? > > Thanks in advance, > > Paul L. Hallberg > CORE Flow Cytometry Facility > Kimmel Cancer Center > Thomas Jefferson University
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