Noncompetitive immunoassays using bifunctional unilamellar vesicles or liposomes

Small unilamellar vesicles (SUVs) functionalized with an enzyme label and with specific ligands for biological molecules are useful as signal enhancement vehicles in the development of enzyme-linked immunoadsorbent assays and other biosensor applications. Bifunctional vesicles were prepared by covalently attaching horseradish peroxidase (HRP) and an antibody to the outside of the lipid bilayer of an SUV. The reaction conditions were optimized to obtain 7-12 antibody molecules and 100-200 HRP molecules per vesicle. The enzyme retained 70-80% of its specific activity after immobilization, and the presence of immobilized proteins on the vesicle surface apparently increased the vesicle stability. To minimize the background signal and maximize the specific signal, the immunoassay protocol was optimized with respect to (1) the type and concentration of blocking agent, (2) the diluents for HRP-antibody-vesicles and sample, (3) the incubation period, and (4) the incubation temperature. The bifunctional vesicles were used in a noncompetitive immunoassay to detect d-dimer, a fibrin dimer formed at the early stages of thrombosis. A second conjugate, HRP-antibody, was prepared, characterized, and used as a control against which to compare the assay using vesicles. The assay results using vesicles led to a detection limit for d-dimer in human plasma 9 times lower than what was achieved using the conventional enzyme-antibody conjugate assay.