Hi All,
Our group is doing some flow cytometry assays using an HLA-A*0201 tetramer presenting an HBV peptide (FLLTRILTI) and we're getting some strange results... We ran tetramer flow on a bunch of patient samples without knowing their HLA haplotype. We found that the tetramer was binding a small proportion of T cells (0.1% or so, as expected) in those patients with history of HBV exposure or vaccination as expected... BUT, upon getting the HLA haplotype data and matching it with the results, we found that the tetramer was binding non-HLA-A2 individuals as well. MFI a bit lower, but definitely positive. Is there any possible explanation for this? No binding of the control peptide was observed, and we also found the same issue with another tetramer presenting a different HBV peptide. We think it mustn't be the tetramers, as they are binding only when the patient has previously experienced the antigen in question.
I'm relatively new to tetramer work, so I don't think this is a dumb question, but I am stumped. Any suggestions/hypotheses would be much appreciated!
Hi Scott,
We have done tetramer work with murine splenocytes, and more recently with patient PBMC samples using tetramers presenting our antigen of interest, generated from the NIH Tetramer Core Facility.
The first things that came to mind are titrations, and incubations.
We did some tests beforehand using 0.25ug, 0.5ug, 1.0ug, 2.5ug, 5.0ug, and 10ug of our test tetramer and control tetramers, as well as incubation times of 30 minutes, 90 minutes, 3 hours, and overnight (18 hours), in both 4°C and 37°C.
From these trials, we saw that under higher tetramer concentrations, we got more non-specific binding of our control tetramer in our samples (seen as a shift of cells that were + for the control tetramer to the right in our gates), and under lower concentrations we lost a good amount of our test tetramer binding (seen a shifting of cells that were + for our test tetramer to the left in our gates). Under the right concentration, incubation time, and incubation condition, we were able to keep our test tetramer + cells, without getting too many control tetramer + cells.
Just a general note when gating for our test tetramer, the corresponding control tetramer gate is set to contain 0.1% of the cells, then that gate is applied to the test tetramer (per NIH Tetramer Core Flow guidance).
Best,
-Ben
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