Comparative study of IgG binding to proteins G and A: nonequilibrium kinetic and binding constant determination with the acoustic waveguide device.
The aim of this work was to measure and compare the binding constants of antibody immunoglobulin G (IgG) to bacterial cell wall proteins, streptococcal protein G and Staphylococcus aureus protein A, using an acoustic wave sensor. Devices, which used shear-horizontal acoustic waves propagating in a waveguide configuration at 108 and 155 MHz, were employed in the detection of apparent IgG binding constants at the solid-liquid interface in the range of 6.7-667 nM IgG. Real-time data during IgG-protein G and IgG-protein A binding yielded apparent association constants of 3.29 x 10(4) and 8.02 x 10(3) M(-1) s(-1) leading to equilibrium constants of 1.13 x 10(8) and 2.90 x 10(7) M(-1), respectively. The measured apparent rate constants are consistent with literature reports of higher affinity of protein G for IgG. Furthermore, protein binding through the Fc region of IgG is suggested to occur below 333 nM, while different mechanisms are suggested to occur above 333 nM. For the first time, nonequilibrium studies of IgG-protein G and A binding at a solid-liquid interface has yielded valuable quantitative kinetic information about binding mechanisms. The promise of this detection method is shown by providing quick determination of binding constants with low sample volumes.
Saha
K
K
Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, United Kingdom.
Bender
F
F
Gizeli
E
E
D000162:Acoustics / Q000295:instrumentation*; D000907:Antibodies, Bacterial / Q000276:immunology; D000915:Antibody Affinity; D001426:Bacterial Proteins / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology*; D013205:Staphylococcal Protein A / Q000276:immunology*
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A