Affinity Interaction between Hexamer Peptide Ligand HWRGWV and Immunoglobulin G Studied by Quartz Crystal Microbalance and Surface Plasmon Resonance

Immunoglobulins (Ig), also referred to as antibodies, act as protective agents against pathogens trying to invade an organism. Human immunoglobulin G (hIgG), as the most prominent immunoglobulin presented in serum and other human fluids, has broad applications in fields like immunotherapy and clinical diagnostics. Staphylococcus aureus Protein A and Streptococcus Protein G are the most common affinity ligands for IgG purifaction and detection. However, drawbacks associated with these two protein ligands have motivated searches for alternative affinity ligands. The hexamer peptide ligand HWRGWV identified from a one-bead-one-peptide combinatorial library synthesized on chromatography resins has demonstrated high affinity and specificity to the Fc fragment of hIgG. A chromatography resin with HWRGWV can purify human IgG (hIgG) from complete minimum essential medium (cMEM) with purities and yields as high as 95%, which are comparable to using Protein A as affinity ligand (4). As a short peptide ligand, HWRGWV can be produced at relatively low costs under good manufacturing practices (GMP) conditions, it is highly robust, less immunogenic and allows for milder elution conditions for the bound antibody (3, 5). Although this short peptide ligand has exhibited promising properties for IgG capture and purification, limited information is available on the intrinsic mechanisms of affinity interaction between the peptide ligand and target protein.

In this study, the affinity interaction between hIgG and peptide ligand immobilized on solid surfaces was studied by quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). Compared with previous methods employed for the peptide characterization, QCM and SPR can provide direct measurements of equilibrium adsorption isotherms and rates of adsorption, allowing a complete kinetic and thermodynamics analyses of the ligand-target interactions.

New methods were developed to modify gold and silica surfaces of QCM and SPR sensors for the immobilization of peptide ligands with low nonspecific binding. The silica surface was first modified by the formation of self-assembling monolayer (SAM) of 3- amino-propyl triethoxy silane as an anchor layer. Short chains of poly(ethylene glycol) (PEG) with Fmoc-protected amino groups at one end and carboxyl groups at the other end were then coupled through the carboxyl terminal to the amino groups on the silane. The short PEG chains served as spacer arms to reduce nonspecific binding to the substrate. The gold surface was modified by a two-component SAM using mixtures of HS(CH2)11(CH2CH2O)6NH2 and HS(CH2)11(CH2CH2O)3OH. The advantage of using a modified silica surface is its relatively higher stability than the SAM on gold during the peptide functionalization step, however the SPR sensors do not work on silica surfaces. In addition, the modification process of the gold surface is relatively simple compared with that of the silica surface. The peptide immobilization process was optimized with silica surfaces and the best conditions were applied for the immobilization on gold surfaces. The results of surface modifications and peptide immobilizations were characterized by various surface analysis techniques including, ellipsometry, contact angle goniometer, chemical force microscopy (CFM), x-ray photoelectron spectroscopy (XPS) and time of flight secondary ion mass spectroscopy (ToFSIMS).

QCM and SPR results indicated that this peptide ligand HWRGWV immobilized on modified silica or gold surfaces has high affinity and specificity to hIgG binding even in a complex medium such as cMEM. Both thermodynamic and kinetic parameters of affinity interaction were obtained by the analysis of QCM and SPR data. Compared with QCM, SPR is more suitable for quantitative analysis of the protein binding, which is essential for the investigation of thermodynamics and kinetics parameters. The maximum binding capacity (4.15 mg m-2) and the dissociation constant (1.83 µM) derived from SPR data are both close to those obtained with chromatography techniques. The association and dissociation rate constants (0.68 m3 mol-1 s -1 and 1.24 s-1 respectively) were acquired for the first time for the affinity binding of IgG on peptide ligand HWRGWV functionalized surface. Although QCM is not as quantitative as SPR, it provides additional information on the status of the adsorbed layers. For instance, the dissipation measurement of QCM indicated that no significant denaturation of adsorbed hIgG occurred during the adsorption process. In addition, it was shown that the peptide ligand immobilized on modified silica surfaces has similar affinity and binding characteristics for IgG adsorption as on modified gold surfaces.

In summary, new surface modification strategies were developed to study the affinity interaction between peptide ligands and target biomolecules. The use of Fc-specific binding peptides on QCM and SPR sensors could result in new devices for IgG concentration determination and also have promise as platforms for the development of immunosensors.

Publication year: 2010
Authors: Fei Sen; Under the direction of Dr. Ruben G. Carbonell

North Carolina State University, Raleigh, North Carolina

Published in: Ph.D Dissertation, 2010, Chemical Engineering


affinity interaction hIgG immunoglobulin immunosensor platform nonspecific binding peptide ligand QCM self-assembling monolayer (SAM) SPR


Other publications