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Help! Question regarding last step of ELISA - (Mar/20/2008 )

I'm using a system where my secondary is biotin-conjugated, and I'm adding streptavadin-AP after, and the substrate is PNPP.
Because of time constraints, I was wondering if after washing out the Step-AP, I could leave the plate overnight (at 4 C) in either PBS or substrate buffer, and add the substrate in the morning.

Anybody ever try this??


I would not extend the PBS incubation overnight. If you cannot do the complete assay in one day, do the binding step overnight, then wash and do the rest of the ELISA the next day.

Better still, amend the assay so you can do the whole thing in one day. You might have to increase the concentration of the binding Ag, or the concentration of the substrate, and test the time it takes to get your standard curve to the required OD.


i incubate the strep-hrp overnight at 4 degrees and it gives reproducible results

hope this helps




I manufacture elisa's and I have added wash buffer (TBS/Tween) in between steps with success. However, I'm not sure I'd do it with the enzyme. Streptavidin-HRP may work, but AP isn't as robust as HRP. If you have any more questions please feel free to email me. I'd be happy to help.

Have a good day.