Specific detection of gastric alpha-antitrypsin by immobilized trypsin on polyHEMA films

Early diagnosis of gastric carcinoma is crucial for maximizing medical treatment efficacy. For the purpose of real time diagnosis ("virtual biopsy") of stomach malignancy we developed a polyHEMA platform capable of capturing human alpha1-antitrypsin precursor (A1AT); a model proteinaceous luminal biomarker. Its specific attachment to the polymeric platform was accomplished by immobilized trypsin, which was linked to the surface of the polyHEMA film by a series of PEG-based spacers. Recognition was enabled by adapting an ELISA-like methodology, using rabbit anti-A1AT and HRP-conjugated anti-rabbit IgG as a secondary antibody. Since this A1AT-sensing platform was designed to be detected by endoscopic means such as a video capsule, its physical stability was tested after casting on top of a polycarbonate surface. It was found that, in contrast to classical ELISA analysis performed on polystyrene plates, A1AT detection was possible only when spacer arms were used to immobilize the capturing moiety, trypsin, with a 7-fold increase in the optical signal and a saturation kinetics dependency upon the concentration of the A1AT biomarker.