How to detach an antibody from cell surface - (Jan/26/2010 )
As I have allready asked in topic title, can you tell me a good way to detach antibodies (after treating my cells with them for a period of time) from the cell membrane. The reason for this is next: i need to see if the treatment has caused the change in antibody production by the cell itself, and in ICH, I need to detect only cell-derived antibodies and not the treatment antibodies, if you know what I mean (since the tretment and produced antibodies are human origine, my secondary antibody recognizes both of them, and that`s the main problem). I have tried with extensive washing with PBS, but that doesn`t help a lot. Do you think that the pronase or trypsin treatment woud help?
surfactant or pH change?
First of all, thank you for answering me . I was thinking to try with 150mM NaCl in bidestilated water, pH 2.5, hope that works. But if it doesn`t, what surfactant did you have in mind?
(that is: Best regards, in my language )
Can you try all 3 treatments in parallel; low pH, enzyme, surfactant (tween 20/sds/triton x100)?
Not sure how would you preserve your cells at harsh conditions required for dissociation of Ab from the cell-bound Ag.
Can you try reduce the treatment Ab to F(ab)2, then use anti-Fc conjugate to detect produced Ab?