title: Universal antibody conjugation to nanoparticles using the Fcγ receptor I (FcγRI): quantitative profiling of membrane biomarkers.
abstract: Antibodies are a class of molecules widely used in bioengineering and nanomedicine for applications involving protein recognition and targeting. Here we report an efficient method for universal conjugation of antibodies to lipid-coated nanoparticles using radially oriented FcγRIs. This method is performed in physiological solution with no additional coupling reagents, thereby avoiding problems with antibody stability and functionality. Coupling to the Fc region of the antibody avoids aggregation and polymerization allowing high yield. In addition, the antibody is oriented perpendicular to the surface so that the binding sites are fully functional. Using this method we demonstrate quantitative profiling of a panel of four membrane-bound cancer biomarkers (claudin-4, mesothelin, mucin-4, and cadherin-11) on four cell lines (Panc-1, MIA PaCa-2, Capan-1, and HPDE). We show that by designing the lipid coating to minimize aggregation and nonspecific binding, we can obtain absolute values of biomarker expression levels as number per unit area on the cell surface. This method is applicable to a wide range of technologies, including solution based protein detection assays and active targeting of cell surface membrane biomarkers.
authors.affiliation: Department of Materials Science and Engineering and ‡Institute for Nanobiotechnology, Johns Hopkins University , 3400 North Charles Street, Baltimore, Maryland 21218, United States.
authors.forename: Chloe
authors.initials: C
authors.lastname: Kim
authors.forename: Justin F
authors.initials: JF
authors.lastname: Galloway
authors.forename: Kwan Hyi
authors.initials: KH
authors.lastname: Lee
authors.forename: Peter C
authors.initials: PC
authors.lastname: Searson
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor / Q000032:analysis; D015820:Cadherins / Q000032:analysis; D045744:Cell Line, Tumor; D062506:Claudin-4 / Q000032:analysis; D058851:GPI-Linked Proteins / Q000032:analysis; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry*; D000090204:Mesothelin; D051379:Mice; D055264:Mucin-4 / Q000032:analysis; D053758:Nanoparticles / Q000737:chemistry*; D010190:Pancreatic Neoplasms / Q000175:diagnosis*; D017452:Receptors, IgG / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D014408:Biomarkers, Tumor; D015820:Cadherins; D062506:Claudin-4; C485701:Fcgr1 protein, mouse; D058851:GPI-Linked Proteins; D018796:Immunoconjugates; C000715528:Msln protein, mouse; D055264:Mucin-4; D017452:Receptors, IgG; D011994:Recombinant Proteins; C086960:osteoblast cadherin; D000090204:Mesothelin