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.

Quantum dot surface engineering: Toward inert fluorophores with compact size and bright, stable emission

“…During coating, some of these anchoring groups unbound to the surface can crosslink QDs, which can be deleterious for most applications [218, 225, 226]. Indeed the coating process is quite different compared with mass action-driven attachment of molecular ligands, as the multidentate binding affinity is so high that the quantity used for coating can be stoichiometric [184].…”

“…Monomeric streptavidin has been used to prevent cross-linking of biotinylated cell-surface proteins but generation of the monomeric streptavidin remains challenging [279]. For antibody conjugation, molecular adaptor proteins such as Protein A/G or Fc Receptor provide unique advantages due to oriented attachment to antibody regions that are not needed for target binding, leaving the target-binding domain extended away from the QD surface, without the need for covalent modification of the antibody [226, 280, 281]. Protein A and G are also oligomers that can bind to multiple proteins, whereas Fc Receptor I is monomeric and binds to a single antibody [226].…”

Recent Advances in the Generation of Antibody–Nanomaterial Conjugates

“…The benefit offered by this approach is that the antibody interaction with the adaptor protein occurs mostly via Fc region, leaving the F ab (antibody-binding site) fully active. [58] These reactions are carried out under physiological conditions and require minimum purification steps, while the antibody retains its stability and functionality. An important property of this approach is that the antibodies can be used interchangeably, and the orientation of the antibodies can be controlled such that the correct presentation to a receptor is preserved.…”

“…Fc receptors are another ideal candidate for multiplexed profiling of individual cellular components. [58] …”

Highly sensitive detection of protein biomarkers via nuclear magnetic resonance biosensor with magnetically engineered nanoferrite particles

“…More detailed protocols regarding the surface modification of NFPs and antibody conjugation with NFPs are indicated in our previous reports. [16][17][18] characterization of nanoferrites…”