I'm doing panning to deplete T&B cells with CD3/CD19 antibodies. CD19 antibodies can remove B cells effectively, however, hamster anti-mouse CD3e can barely adsorb T cells. Both antibodies were marked for FC use. Is it possible that the binding affinity of FC antibody was too low for panning? Or, just the antibodies have lost effectiveness? What kind of antibodies are suitable for panning? Can someone give some advice?
Without knowing how you have tried your panning here are somethings that you should be aware of.
Not every antibody that binds CD3 will work for panning.
How much antibody is actually on the surface will affect panning efficiency. Try a few different coating concentrations to optimize the antibody. It is usualy somewhere between 0.1 to 1 ug/ml in PBS that will optimally coat a polystyrene surface.
The panning conditions may not be optimal. How much Mg and Ca are in your solution. Try panning in PBS without Ca and Mg. This will limit capping off of crosslinked antigen (i.e. CD3). Also the time and temperature are important and may need to be optimized. Lower temp may also prevent capping of crosslinked antigen.
Hope this helps some.
Thanks a lot for the help! In deed, I've tried 2.5 ug/ml in PBS or 0.05 M Tris(pH 9.5). For PBS, I incubated at 4°C overnight, while for Tris Buffer, I did at room temperature for 1.5 hrs. CD19 antibody was done with the same conditions and it worked well under both conditions. The antibody I use for CD3 is anti-mouse CD3e, clone 145-2C11, from biolegend(Cat# 100302). It's said this antibody is suitable for FC(quality tested)
IHC, IF, IP and WB(the latter four, reported in the literature). Can someone tell me if this antibody can be used for Panning? Thanks a lot!
Hi,
maybe this is a useful tool?!
https://www.miltenyibiotec.com/~/media/Images/Products/Import/0001200/IM0001270.ashx
I think CD90.2 works very good in mice, not like CD3...
Hope it helps.
Cheers
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