title: Mechanism of immobilized protein A binding to immunoglobulin G on nanosensor array surfaces.
abstract: Protein A is often used for the purification and detection of antibodies such as immunoglobulin G (IgG) because of its quadrivalent domains that bind to the Fc region of these macromolecules. However, the kinetics and thermodynamics of the binding to many sensor surfaces have eluded mechanistic description due to complexities associated with multivalent interactions. In this work, we use a near-infrared (nIR) fluorescent single-walled carbon nanotube sensor array to obtain the kinetics of IgG binding to protein A, immobilized using a chelated Cu(2+)/His-tag chemistry to hydrogel dispersed sensors. A bivalent binding mechanism is able to describe the concentration dependence of the effective dissociation constant, KD,eff, which varies from 100 pM to 1 μM for IgG concentrations from 1 ng mL(-1) to 100 μg mL(-1), respectively. The mechanism is shown to describe the unusual concentration-dependent scaling demonstrated by other sensor platforms in the literature as well, and a comparison is made between resulting parameters. For comparison, we contrast IgG binding with that of human growth hormone (hGH) to its receptor (hGH-R) which displays an invariant dissociation constant at KD = 9 μM. These results should aid in the use of protein A and other recognition elements in a variety of sensor types.
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
authors.forename: Justin T
authors.initials: JT
authors.lastname: Nelson
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
authors.forename: Sojin
authors.initials: S
authors.lastname: Kim
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
authors.forename: Nigel F
authors.initials: NF
authors.lastname: Reuel
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Salem
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
authors.forename: Gili
authors.initials: G
authors.lastname: Bisker
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
authors.forename: Markita P
authors.initials: MP
authors.lastname: Landry
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Kruss
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Barone
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
authors.forename: Seonyeong
authors.initials: S
authors.lastname: Kwak
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Strano
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000295:instrumentation* / Q000379:methods*; D005453:Fluorescence; D019382:Human Growth Hormone / Q000737:chemistry; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D046228:Microarray Analysis; D037742:Nanotubes, Carbon / Q000737:chemistry; D011485:Protein Binding; D013205:Staphylococcal Protein A / Q000737:chemistry*; D013499:Surface Properties
chemical_list: D055767:Immobilized Proteins; D007074:Immunoglobulin G; D037742:Nanotubes, Carbon; D013205:Staphylococcal Protein A; D019382:Human Growth Hormone