Antibodies will bind denatured protein (urea-treated one) if you have lineal epitopes but normally these are conformationals. If antibodies are polyclonal antibodies, maybe part of them will be able to bind a denatured protein, but no monoclonal antibodies just if these were prepared against a lineal epitope.

The second question is so confuse. Do you want to remove the coated antibodies?.

Samar, question 2 is easy to answer if it works for your antibody: just try it, you may test for success with anti species peroxidase conjugate. In general, I would not expect complete removal of an antibody once it has been adsorbed to a plastic surface,

Dear Ana,

Thanks for your response.

No, I don't want to remove the coat.

let me explain my question further, if I want to use urea in a sandwich ELISA to reduce the cross-reactivity of the primary antibody to a serum component (Ag1) rather than antigen of interest (Ag2), which already has been demonstrated using purified antigens, coated onto plate. Is using urea going to wash away our capture antibody that being coated onto plate? because I'm trying now to asses the effect of using Urea on the specific binding between the analyte and the capture Ab (monoclonal) by doing the following

Coat the plate with capture Ab >>> Wash PBST >>> incubation of the purified antigen (Ag1+Ag2) >> wash PBST >> Urea (2,4,8M for 10, 30 min and 1h) >> wash PBST>> HRP conjugated polyclonal Ab toward Ag1 & Ag2.

I'm not sure if it was the correct way for assessing that effect. I can see changes in ODs upon using urea but I cannot be sure why!

Is it possible that Urea wash off the capture? OR/AND that was because of urea disfiguration of epitopes, so the polyclonal couldn't bind them as it would be for untreated -Ag. Therefore, I am not able to tell if changes are really due to disassociation between capture Ab and Ag.

N.B. this ELISA is to distinguish Ag2 from Ag1 (which are different forms of the same protein), and both of them can be captured by our capture monoclonal.

Urea, in the final assay, are intended to be added after the step of incubating the primary Ab.

Nunc MaxiSorp microplates, were used

Hoping that not more confusing as it is for me :) ....... please let me know what do you think!

Currently antibodies do not recognize denatured and native antigens in the same way. For detecting the urea effect if any, try different urea concentratons (2, 4 and 8 M). I think 3 M urea is more than enough for denaturation. 

please find the link. i think that will be helpful for you to get the answer.

http://www.sciencedirect.com/science/article/pii/0019279164900291

For question 1, 8M urea-treated protein antigen is able to be coated onto the microplate, and the linear epitope on the protein would be recognized by pAb, or MAb if it recognize the linear epitope.

I don't fully understand question 2. maybe urea can wash off coated antibody or maybe it can't, but can leads to conformational changes to the coated antibody by denaturing it.

Good luck.

Urea has bee used to check avidity of antibodies to antigen. Up to 8 M urea can be used and the antigen is not removed from the plate. I  used frequently to check my monoclonal antibodies for the best avidity to the antigen.

See the following article for reference from others.

Use of an Immunoglobulin G Avidity Test To Discriminate between Primary and Secondary Dengue Virus Infections

J Clin Microbiol. 2004 Apr; 42(4): 1782–1784.

Janos

As someone said above, you can have antibodies against lineal epitopes.

You can remove urea from coated plates, I' ve done that without problem.