Protein G-liposomal nanovesicles as universal reagents for immunoassays

Abstract: To improve the antigen-binding activity of liposome-coupled antibodies and to develop universal liposomal nanovesicles for immunoassays, protein G was conjugated to dye-loaded liposomal nanovesicles for the preparation of immunoliposomes. Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC), a heterobifunctional cross-linker, was used to modify protein G for conjugation to the liposomal nanovesicles. Liposome immunosorbent assays were used to evaluate the binding ability of protein G after sulfo-SMCC modification, to optimize the protein G density on the liposome surface and to determine the amount of IgG binding to the protein G-liposomal nanovesicles. Test strips coated with a narrow zone of antibodies were used to show the successful conjugation. Immunomagnetic beads were used to demonstrate the feasibility of protein G-tagged universal liposomal nanovesicles for immunoassays. Results indicate that the Fc-binding capacity of protein G decreased by only 5.3% after sulfo-SMCC modification. Antibodies were easily conjugated to universal protein G-liposomal nanovesicles in 30min. The conjugates (protein G-immunoliposomes) were successfully used in immunomagnetic bead assays for the detection of Escherichia coli O157:H7 with a detection limit of approximately 100CFU/ml. This work demonstrated that protein G-liposomal nanovesicles are a successful universal reagent for easily coupling antibodies in an active orientation on the liposome surface for use in immunoassays. [Copyright &y& Elsevier]