Originally Posted by iceman967
Thanks so much for the help. I will try that this week. But just out of curiosity, why would a higher capture antibody concentration lead to a higher signal, independent of the antigen concentration. Could that mean that the secondary is binding to the capture as well?
Higher capture antibody is used to coat the wells and leave as little empty space available for non-specific binding.
Your assay is sound but you really need excellent blocking (and a working antibody pair and positive control protein) to get good results.
Good blocking will come easier by coating the well with a lot of capture antibody and then blocking with a great blocking solution.
(both the 10ug/ml concentration and startingblock recommendations are to save you time from optimizing one variable/step at a time)
Secondary antibody won't bind to the capture antibody, but it could (and may currently be doing so) bind to empty polystyrene space (polystyrene naturally and strongly binds protein) thus leading to false signal; which you'll see in the "blanks or neg ctrls".