標題: Probing the binding kinetics of proinflammatory cytokine-antibody interactions using dual color fluorescence cross correlation spectroscopy
作者: Wu, Chia-Yan
Huang, Chuan-Keng
Chung, Chao-Yu
Huang, I-Ping
Hwu, Yeukuang
Yang, Chung-Shi
Lai, Yiu-Kay
Lo, Leu-Wei
Chiang, Su-Yu
應用化學系
Department of Applied Chemistry
公開日期: 2011
摘要: 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.
URI: http://hdl.handle.net/11536/26003
http://dx.doi.org/10.1039/c0an00995d
ISSN: 0003-2654
DOI: 10.1039/c0an00995d
期刊: ANALYST
Volume: 136
Issue: 10
起始頁: 2111
結束頁: 2118
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