Syntheses of antigens conjugated with 3-methoxy-4-hydroxyphenylglycol by Mannich reaction for enzyme immunoassay

We have been involved in developing a specific enzyme immunoassay (EIA) for an endogenous d-3-methoxy-4-hydroxyphenylglycol (d-MHPG) which will be a diagnostic marker for mental diseases such as depression. Various antigens were prepared by Mannich reaction of the haptens with carriers and screened to make a good condition for EIA and to elicit the most specific antibody. A model compound of MHPG with 8-aminocaprylic acid by Mannich reaction was synthesized and identified by 1H- and 13C-NMR spectroscopy. Even the hapten structure of the antigen was confirmed by 13C-NMR. The model compound condensed with the carrier protein was not so immunogenic for mice. dl–MHPG conjugated with human serum albumin (HSA) was the strongest immunogenic among those conjugated with bovine serum albumin, ovalbumin (OVA) and HSA. As the adsorbent for EIA, dl-MHPG–OVA was selected to evade the nonspecific binding of the carrier. d-MHPG–HSA was much better in chiral and structural specificity than dl- or l-MHPG–HSA. The other antigens such as d-MHPG-multiple antigenic peptide and 4-sulfo-dl-MHPG–HSA were not suitable to produce specific antibody. Thus, dl-MHPG–HSA was decided to be the best antigen to prepare a monoclonal antibody.