Immunoreactivity of synthetic peptide

Hello all,

I am working in an immunology/micro lab investigating a certain sexually transmitted infection. We have done epitope mapping and determined the amino acid sequences of the various epitopes they recognize. We are working toward development of a diagnostic and used the epitope information to synthesize a ~25KDa peptide containing six of the epitopes that both men and women see. However they peptide is not reacting very well (if at all) by immunoblot or ELISA. Even throwing straight hybridoma supernatant of a monoclonal antibody to the most reactive of the epitopes still gives no signal. We have used two different secondary antibodys conjugated to HRP and AP and get similar results. We have changed the blocking buffer, antibody buffer, time and temperature of the primary and secondary incubations; pretty much I am running out of ideas.

Any insight into what may be the issue here? The only thing I can think of is taking the purified peptide and getting it sequenced just to make sure its there.

Any help would be greatly appreciated.

Thanks,

-PJ