sandwich elisa - how to increase O.D. (Aug/12/2008 )
Hi all,
I was doing Sandwich elisa with antibody coating first but I am not getting good O.D. irrespective of antibody i am using
but the detecting antibodies give good O.D. in indirect elisa
i think it is the way i am coating the antibdy
the protocol i am following is coating 1000ng of antibody in 100ul of PBS at 37C 2hrs or 4C overnight
can someone tell me if there is another better way to coat antibody for sandwich elisa
thanking you in advance
Leelaram
use carbonate/bicarbonate buffer (pH7.4) instead of PBS for coating buffer.
I use carnonate/bicarbonate buffer ph=9.6 instead of coating buffer
I think u can do a little check by coating with PBS(I think the ph should be 7.4) and carbonate/bicarbonate buffer(ph9.6) to see from which one u can get the higher signal.
Besides, different blocking buffer and different titre and kinds of abs should give different signal.
sorry made a mistake... carbonate/bicarbonate buffer should be pH 9.6
try with both PBS and carbonate and use secondary abs anti-"your coating ab's specie" to detect how much ab you have onto the plate. try also to decrease the concentration of the abs