Preparation and Characterization of Bifunctional Unilamellar Vesicles for Enhanced Immunosorbent Assays

Matthew A. Jones, Peter K. Kilpatrick, and Ruben G. Carbonell

Department of Chemical Engineering, North Carolina State University, Raleigh, North Carolina 27695-7904

Small unilamellar phospholipid vesicles with covalently attached biotin and horseradish

peroxidase (HRP) were prepared and characterized in terms of hydrodynamic diameter, amount and activity of immobilized enzyme, and number of biotin molecules on the outer vesicle surface. In addition, the specific adsorption of these bifunctional vesicles and commercially available biotin-labeled horseradish peroxidase (B-HRP) to anti-biotin antibody (ABA) coated polystyrene microliter plate wells was examined. At low antibody surface densities, the signal (DA/min) generated by the vesicles adsorbed to the surface was approximately 100 times higher than the signal generated by B-HRP. It was also found that the biotin-conjugated vesicles were able to compete effectively with free biotin in solution for surface ABA sites. These results indicate that this type of vesicle may be used in competitive and sandwich-type enzyme-linked immunoassays to improve the detection limits, increase the signal, and decrease the reaction time necessary to detect a given analyte concentration in solution.