Probing the binding kinetics of proinflammatory cytokine-antibody interactions using dual color fluorescence cross correlation spectroscopy

Dual color fluorescence cross correlation spectroscopy (FCCS) was used to investigate quantitatively the binding kinetics of tumor necrosis factor (TNF alpha) with TNF alpha antibody (anti-TNF alpha) following fluorescent labeling. Through the analysis of the auto correlation curves of fluorescence correlation spectroscopy (FCS), diffusion coefficients of 100.06 +/- 4.9 mu m(2) s(-1) and 48.96 +/- 2.52 mu m(2) s(-1) for Alexa488-TNF alpha and Atto647N-anti-TNF alpha were obtained. In addition, the calculated hydrodynamic diameters of the Alexa488-TNF alpha and Atto647N-anti-TNF alpha 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 TNF alpha and the anti-TNF alpha 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) x 10(4) M(-1) s(-1) and (1.53 +/- 0.19) x 10(-3) s(-1) while the corresponding dissociation constant (K(d)) at 25 degrees C was (1.36 +/- 0.10) x 10(-7) M. We believe this is the first report on the binding kinetics for TNF alpha-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.