Talanta 67 (2005) 205–211 Protein G-liposomal nanovesicles as universal reagents for immunoassays Chien-Sheng Chen a , Antje J. Baeumner b , Richard A. Durst a,∗ a Department of Food Science & Technology, Cornell University, Geneva, NY 14456, USA b Department of Biological & Environmental Engineering, Cornell University, Ithaca, NY 14853, USA Received 6 January 2005; accepted 16 February 2005 Available online 17 March 2005 Abstract To improve the antigen-binding activity of liposome-coupled antibodies and to develop universal liposomal nanovesicles for immunoas- says, 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 30 min. 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 100 CFU/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. © 2005 Elsevier B.V. All rights reserved. Keywords: Protein G; Liposome; Nanovesicle; Universal reagent; Immunoassay 1. Introduction Liposomal nanovesicles, i.e., liposomes, are spher- ical vesicles consisting of phospholipid bilayers sur- rounding an aqueous cavity. Because each liposomal nanovesicle can contain up to several million fluorescent dye molecules, thereby providing greatly enhanced sig- nals, antibody-tagged liposomal nanovesicles (immunoli- posomes) have been successfully used as reporter parti- cles in immunoassays [1–4]. The strategies for conjugat- ing antibodies to liposomal nanovesicles involve mostly covalent binding using cross-linking molecules, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N- ∗ Corresponding author. Tel.: +1 315 787 2297; fax: +1 315 787 2397. E-mail address: rad2@cornell.edu (R.A. Durst). succinimidyl-S-acetylthioacetate (SATA), succinimidyl-4- (N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) and succinimidyl-4-(p-maleimidophenyl)butyrate (SMPB) [5–8]. The noncovalent biotin–(strept)avidin coupling method also involves the biotinylation of antibodies [9]. These methods generally require the use of the amino groups on the antibody. This approach, however, is limited because most antibodies contain randomly distributed amino groups, leading to multiple attachment sites. The random nature of this attachment can cause some of the conjugated antibod- ies to lose antigen-binding activity due to direct chemical modification or steric hindrance of the antigen-binding site [10,11]. To improve the antigen-binding activity of liposome- tagging antibodies, IgG Fc-binding proteins, like protein A or protein G, can be used to couple antibodies to liposomal 0039-9140/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2005.02.018