Assay Development

We have access to a purified tumor associated peptide along with polyclonal antibody to develop an ELISA. We would like a detailed protocol to develop an ELISA quickly using a microtiter plate format. Would like specific recommendations regarding how to coat the plates with antibody to insure that it sticks and suggestions for developing an ELISA as opposed to an IRMA.

You've not given any clues as to why your challenge is anything out of the ordinary. Why are you mentioning to a immunoradiometric assay (IRMA)? Do you have a particular problem with sensitivity? A particular problem with protein binding to your wells?

How big is your peptide?
Is your antibody working?

What are you trying to achieve with your assay - if you've already purified your protein and are trying to quantify the protein there may be better approaches. Or are you planning to use your purified protein to calibrate an ELISA?

Members of the forum will be able to help if best if you ask a specific question.

The protein I have in mind is a 9.5kD ribosomal subunit zinc-finger protein that is passively secreted from cells. The primary antibody is from rabbits.
We are having problems with the antibody sticking to microtiter plates in order to develop a sandwich type ELISA. Any suggestions about plate coating?