I am currently trying to create an indirect ELISA protocol for measuring the competitive adsorption of human albumin, gamma globulin, and fibrinogen onto various polyethylene samples. The samples are rather large (20X15X3 mm) and, in the first step, require incubation with 5-10 ml of protein solution (Albumin - 4000 ppm; gamma globulin - 1000 ppm; and fibrinogen - 300 ppm). In the second step, the samples (after washing) will be incubated with goat anti-human antibodies for albumin, gamma globulin, or fibrinogen. However, I do not have very much experience with ELISA (I have performed ELISA kit experiments before, but nothing more), and I am unsure in what concentrations I should prepare the primary antibody solutions. In references I have seen people quote concentrations ranging fro 1/400 to 1/5000. However, if I want the antibodies to be in excess of the attached protein, wouldn't I need to use a concentration greater than 1/400? Especially if my initial protein solution has a concentration of 4000 ppm (using albumin as an example)? Any help or guidance that you can provide would be very much appreciated. Thank you.
Edited by Water Bear Minimum, 04 January 2012 - 09:21 PM.