My question is with regards to the usability of the indirect ELISA in measuring protein concentration. Suppose we want to quantify the concentration of a protein. The indirect ELISA method would require one to coat the bottom of the well with protein antigen and wash off unbound antigens.
My question is that wouldn't washing off unbound proteins mean you are 'diluting' the actual protein concentration? Your resulting colour intensity will not reflect the actu protein concentration. Thanks!