Noncompetitive Immunoassays Using Bifunctional
Unilamellar Vesicles or Liposomes
Anup K. Singh, Peter K. Kilpatrick, and Ruben
G. Carbonell
Department of Chemical Engineering,
Small unilamellar vesicles (SWs) 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.