PREPARATION AND CHARACTERIZATION OF BIFUNCTIONAL BIOPOLYMERS FOR RECEPTOR-BASED LIPOSOMAL IMMUNOSENSING

In this study, we prepared bifunctional biopolymers for development of a novel liposomal immunosensing element. These biopolymers were produ ced such that a rat monoclonal antibody fragment Fab' was linked to a cardiac protein Troponin I (TnI) peptide by a cross-linking reagent, o -phenylenedimaleimide (o-PDM) or N-sucinimidyl 3-(2-pyridyldithio)prop ionate (SPDP). The biopolymer formation yields were approximately 10% for Fab-TnI(Mal) and 30% for Fab-TnI(SPDP). Molar ratios of Fab' to SP DP or o-PDM and conjugated Fab' to TnI peptide and conjugation pH have considerable effects on the biopolymer yield. Purification of these b iopolymers was achieved by employing size-exclusion HPLC. These biopol ymers can bind to receptor channels on one end, while the peptide end can be recognized by an anti-TnI antibody serving as a protein linker to block the channels in the immunosensing element. Then reactions may be used where free analyte competes for cross-linker binding sites wh ereby channels are rendered active. Characterization of purified biopo lymers was performed using gel electrophoresis, ELISAs, and a BIAcore instrument. Furthermore, results of real-time biospecific interaction experiments with use of the BIAcore show that competition binding reac tions of free TnI peptide occurred in this new immunosensing design. T he binding activities of these two biopolymers are slightly different.