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 conjugated with thousands of fluorescein molecules and 10-20 monoclonal antibodies per liposome. These bifunctional liposomes were used in a direct (sandwich-type) immunoassay for the detection of thromboembolic disorders by assaying for d-dimer. D-dimer is the final and the smallest proteolytic product in the degradation of cross-linked fibrin by the plasma protein plasmin. The immunoassay using liposomes was compared to a conventional immunoassay that uses a fluor-antibody conjugate. The liposomes, by virtue of having thousands of fluorophores coupled to one liposome in contrast to one or a few reporter molecules in the conventional fluor-antibody conjugate, performed better on two counts: (1) they lowered the detection limit by a factor of 120 and (2) they provided a 1 order of magnitude amplification in signal. The minimum detectable concentration (MDC) of d-dimer was 5.6 ng/mL with the liposomal assay 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. Prog. 1995, 11, 333-341) in an enzyme immunoassay developed using liposomes. These results demonstrate the potential of liposomes in lowering detection limits and increasing the sensitivity of immunoassays.
Singh
A K
AK
Department of Chemical Engineering, North Carolina State University, Raleigh 27695-7905, USA.
Kilpatrick
P K
PK
Carbonell
R G
RG
D000911:Antibodies, Monoclonal / Q000276:immunology*; D005338:Fibrin Fibrinogen Degradation Products / Q000032:analysis*; D016650:Fluorescein-5-isothiocyanate; D007118:Immunoassay / Q000379:methods*; D008081:Liposomes
D000911:Antibodies, Monoclonal; D005338:Fibrin Fibrinogen Degradation Products; D008081:Liposomes; C036309:fibrin fragment D; D016650:Fluorescein-5-isothiocyanate