Application of Antibody and Fluorophore‐Derivatized Liposomes to Heterogeneous Immunoassays for D‐dimer

Abstract

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.

Biosensors for Cancer Biomarkers

“…Liposomes were created to be characterized by determining the homogeneity of the lipid results were obtained from each liposome(eq 1). aL per lipid head group surface area, d is the hydrodynamic radius, t the thickness of the lipid bilayer (Singh et al, 1996).…”

Bulk fluorescence light blockers to improve homogeneous detection in capillary-waveguide fluoroimmunosensors

“…The labeled antibody was isolated from unreacted FITC molecules using a PD-10 column equilibrated with a 0.1 mol l −1 phosphate buffered saline, pH 7.4. The number of fluorescein molecules per antibody, determined according to Singh et al (1996), was approximately 3.5.…”

Improved Performance of Antigen-HRP Conjugate-based Immunoassays after the Addition of Anti-HRP Antibody and Application of a Liposomal Chemiluminescence Marker

“…fluorescent molecules or enzymes. [2][3][4][5][6][7][8] Enzymes such as glucose oxidase, 9 alkaline phosphatase, 10 horseradish peroxidase (HRP) 3,7,[11][12][13][14][15] and β-galactosidase, 16 have been applied as labels, either conjugated to the outside of the lipid bilayer of liposome or encapsulated into liposomes.…”