Dual color fluorescence cross correlation spectroscopy (FCCS) was used to investigate quantitatively the binding kinetics of tumor necrosis factor (TNFalpha) with TNFalpha antibody (anti-TNFalpha) following fluorescent labeling. Through the analysis of the auto correlation curves of fluorescence correlation spectroscopy (FCS), diffusion coefficients of 100.06 +/- 4.9 mum(2) s(-1) and 48.96 +/- 2.52 mum(2) s(-1) for Alexa488-TNFalpha and Atto647N-anti-TNFalpha were obtained. In addition, the calculated hydrodynamic diameters of the Alexa488-TNFalpha and Atto647N-anti-TNFalpha 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 TNFalpha and the anti-TNFalpha 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 TNFalpha-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.
