capture antibody and law of mass action - (Mar/19/2014 )

I am student and I work on ELISAs trying coater Abs at the bottom of a plate but I want to know if there is a real impact of the law of mass action for the recognition of the antibody coated Abs plate. (I'm french so sorry for my language)

Are you asking how the law of mass action impacts the binding of the coating antibodies to the ELISA plate?  While there is an equilibrium to most binding events, the dissociation of antibodies from the high-binding plastic plates is generally slow so the impact should be minimal.

Yes I'm asking this. Because, we don't know the concentration of our Abs.

We know only that concentration is of the order of mg/mL.

are your antibodies purified?  If they are, a spec reading at A280 can give you the mg/mL value of your antibody (divided by the appropriate extinction coefficient).  If they are not purified then you will have other problems as coating a plate with a mixture of proteins will dramatically reduce the sensitivity of your ELISA.

Our antibodies aren't really purified. In fact, we know that there aren't other proteins but there are surely other antibodies not specific of the target antigens. Thank you for your answers so fast.