ELISA controls - (Oct/21/2004 )

I need some help in figuring out what exactly to use as controls in my Elisa setup. What is the proper control for background determination?I have done it a number of ways - no Ag + primary and secondary Ab, Ag + secondary with no primary , etc. Also, are you supposed to subtract these background readings from your samples of interest or do you just run these controls to ensure their ODs aren't too high. Or maybe this is not necessary at all? Any help would be greatly appreciated!!

it depends.
for antigen capture ELISA we use the 1st (capture) AB, no antigen, 2nd (detection) AB method for negative contols. the OD measurement of these contol wells is subtracted from every other well on the same plate as background.

for antibody capture ELISA, we use antigen, control AB, 2nd AB. the control antibody e.g. is for the determination of serum antibody titers after immunisation: a pre-immune serum or serum from a not or only mock immunised animal. as above the OD measurement of these contol wells is subtracted from every other well on the same plate as background.

mike