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 signa l enhancement vehicles in the development of enzyme-linked immunoadsor bent assays and other biosensor applications. Bifunctional vesicles we re prepared by covalently attaching horseradish peroxidase (HRP) and a n 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 speci fic activity after immobilization, and the presence of immobilized pro teins on the vesicle surface apparently increased the vesicle stabilit y. To minimize the background signal and maximize the specific signal, the immunoassay protocol was optimized with respect to (1) the type a nd 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 stag es of thrombosis. A second conjugate, HRP-antibody, was prepared, char acterized, and used as a control against which to compare the assay us ing vesicles. The assay results using vesicles led to a detection limi t for d-dimer in human plasma 9 times lower than what was achieved usi ng the conventional enzyme-antibody conjugate assay.