ELISA diluent options - (May/06/2007 )
Quick question for the ELISA-heads out there.
I'm having some trouble replicating an assay with my own reagents. The one big stumbling-block at the moment is the choice of sample diluent buffer. I've tried PBS, PBS/1% BSA, PBS/0.025% or 0.05% Tween20, and TBS with BSA. I've also seen diluents like 5% Tween in PBS (which sounds a bit extreme to me, but if it works...)
Does anyone have any other options for diluents?
TBS + 5% skim milk
Using PBS + 1%BSA + 0.05% Tween20 has always worked for me.
I had produced a monoclonal antibody in my lab, but when I use it in ELISA for coating or as 1st Ab I see cross reactivity with the detection Ab. I would like to know if you can help me solving this problem. I also found a new buffer (called LowCross-Buffer) that is recommended to reduce interferences in this assays, and I would like to know if someone already use it before.
Thanks in advance
Have you tried titrating out your detection ab? I have found that cross-scrubbed secondary detection antibodies from Jackson work really well. Make sure that if your monoclonal is mouse that your detection is adsorbed against rat as well as mouse.