I am using soluble anti-CD3 (clone UCHT1) and anti-CD28 antibodies to activate T cells in whole blood. The activation is followed by detection of the different cell types with flow cytometry, where among others, I use anti-CD3 (clone SK7). Since I started adding the anti-CD3 in soluble form I cannot find the CD3+ population in my activated samples (never had this problem when using plate bound anti-CD3 for the activation). I read there is evidence that the SK7 and UCHT1 monoclonal antibodies might cross-block binding, suggesting recognition of overlapping epitope.
Has any of you ever come across that same problem of cross-block binding of UCHT1 with other clones and do you have any suggestion what I can do and of another antibody clone to use to detect my CD3+ cells?
Thank you very much for your help!
I've never used the SK7 clone but have used OKT3 as a CD3 agonist and UCHT1 to detect. Although I haven't done them at the same time so I don't know if they block each other. Either way, OKT3 is an awesome clone for stimulation so you could give that a try
Hi Georgia,
In my experiment, I used CD4 isolated kit for obtaining CD4+ T cells and used soluble form anti-CD3 Ab (OKT3 clone) and anti-CD28 Ab to stimulate the T cells. Then used anti-CD4 and antibody against other activated marker, like PD-1 or others, for detecting the activated population of T cells.
If you want to know whether there is blocking effect between these two Abs, competition assay may answer your question.
Hope this is helpful.
Weber
Hello Georgia,
Have you tried indirect staining? I think that it should solve the problem.
The problem is the UCHT1 clone is bound to the CD3 molecule even after stimulation. Hence there are no epitopes for your SK7 clone to bind to (as the epitopes overlap). If you add a secondary antibody to the UCHT1 clone following permeabilization (CD3 gets internalized during stimulation), you will be able to visualize CD3 population.
The reason why you didn't have a problem with plate bound CD3 --- during washing, the cells come off and most of your antibody is still bound to the plate. Hence the CD3 epitope is available for a different CD3 antibody.
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