Abstract
Dual color fluorescence cross correlation spectroscopy (FCCS) was used to investigate quantitatively the binding kinetics of tumor necrosis factor (TNFα) with TNFα antibody (anti-TNFα) following fluorescent labeling. Through the analysis of the auto correlation curves of fluorescence correlation spectroscopy (FCS), diffusion coefficients of 100.06 ± 4.9 μm 2 s -1 and 48.96 ± 2.52 μm 2 s -1 for Alexa488-TNFα and Atto647N-anti-TNFα were obtained. In addition, the calculated hydrodynamic diameters of the Alexa488-TNFα and Atto647N-anti-TNFα were approximately 4.89 ± 0.24 nm and 9.99 ± 0.52 nm, respectively, which agrees with the values of 5.20 ± 1.23 nm and 9.28 ± 0.86 nm for the native TNFα and the anti-TNFα as determined from dynamic light scattering measurements. For the binding kinetics, association (k on ) and dissociation (k off ) rate constants were (1.13 ± 0.08) × 10 4 M -1 s -1 and (1.53 ± 0.19) × 10 -3 s -1 while the corresponding dissociation constant (K d ) at 25 °C was (1.36 ± 0.10) × 10 -7 M. We believe this is the first report on the binding kinetics for TNFα-antibody recognition in the homogeneous phase. Using this technology, we have shown that controlled experiments can be performed to gain insight into molecular mechanisms involved in the immune response. © 2011 The Royal Society of Chemistry.