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

Chia-Yan Wu; Chuan-Keng Huang; Chao-Yu Chung; I-Ping Huang; Yeukuang Hwu; Chung-Shi Yang; Yiu-Kay Lai; Leu-Wei Lo; Su-Yu Chiang

doi:10.1039/C0AN00995D

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

Chia-Yan Wu,^a Chuan-Keng Huang,†^b Chao-Yu Chung,†^d I-Ping Huang,^c Yeukuang Hwu,^e Chung-Shi Yang,^f Yiu-Kay Lai,^c Leu-Wei Lo*^a and Su-Yu Chiang*^d

Author affiliations

* Corresponding authors

^a Division of Medical Engineering Research, National Health Research Institutes, Zhunan, Miaoli 35053, Taiwan ROC
E-mail: lwlo@nhri.org.tw
Fax: +886-37-586440
Tel: +886-37-246166 x37115

^b Department of Applied Chemistry, National Chiao Tung University, Hsinchu 30010, Taiwan ROC

^c Institute of Biotechnology, National Tsing Hua University, Hsinchu 30076, Taiwan ROC

^d National Synchrotron Radiation Research Center, 101, Hsin Ann Road, Science-Based Industrial Park, Hsinchu 30076, Taiwan ROC
E-mail: schiang@nsrrc.org.tw
Fax: +886-3-5789816
Tel: +886-3-5780281 x7315

^e Institute of Physics, Academia Sinica, Taipei 11529, Taiwan ROC

^f Center for Nanomedicine Research, National Health Research Institutes, Zhunan, Miaoli 35053, Taiwan ROC

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 μm² s⁻¹ and 48.96 ± 2.52 μm² 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) × 10⁴ 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.

Analyst

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

Abstract

Article information

Download Citation

Permissions

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

Social activity

Search articles by author

Spotlight

Advertisements