APPLICATION OF ANTIBODY AND FLUOROPHORE-DERIVATIZED LIPOSOMES TO HETEROGENEOUS IMMUNOASSAYS FOR D-DIMER

Small unilamellar liposomes comprised of cholesterol and phospholipids , in which one of the lipids is labeled with a fluorophore, have been covalently functionalized with antibodies. The liposomes were conjugat ed with thousands of fluorescein molecules and 10-20 monoclonal antibo dies per liposome. These bifunctional liposomes were used in a direct (sandwich-type) immunoassay for the detection of thromboembolic disord ers by assaying for d-dimer. D-dimer is the final and the smallest pro teolytic product in the degradation of cross-linked fibrin by the plas ma protein plasmin. The immunoassay using liposomes was compared to a conventional immunoassay that uses a fluor-antibody conjugate. The lip osomes, by virtue of having thousands of fluorophores coupled to one l iposome in contrast to one or a few reporter molecules in the conventi onal fluor-antibody conjugate, performed better on two counts: (1) the y lowered the detection limit by a factor of 120 and (2) they provided a 1 order of magnitude amplification in signal. The minimum detectabl e concentration (MDC) of d-dimer was 5.6 ng/mL with the liposomal assa y as compared to an MDC of 674 ng/mL with conventional fluor-antibody conjugate. The results of fluorescence assays were also compared with the results obtained by Singh et al. (Biotechnol. Frog. 1995, 11, 333- 341) in an enzyme immunoassay developed using liposomes. These results demonstrate the potential of liposomes in lowering detection limits a nd increasing the sensitivity of immunoassays.