title: Probing the binding kinetics of proinflammatory cytokine-antibody interactions using dual color fluorescence cross correlation spectroscopy.
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.
authors: Division of Medical Engineering Research, National Health Research Institutes, Zhunan, Miaoli, Taiwan ROC.
authors: Chia-Yan
authors: CY
authors: Wu
authors: Chuan-Keng
authors: CK
authors: Huang
authors: Chao-Yu
authors: CY
authors: Chung
authors: I-Ping
authors: IP
authors: Huang
authors: Yeukuang
authors: Y
authors: Hwu
authors: Chung-Shi
authors: CS
authors: Yang
authors: Yiu-Kay
authors: YK
authors: Lai
authors: Leu-Wei
authors: LW
authors: Lo
authors: Su-Yu
authors: SY
authors: Chiang
mesh_terms: D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D003116:Color; D005456:Fluorescent Dyes / Q000737:chemistry; D057446:Hydrodynamics; D007700:Kinetics; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000737:chemistry* / Q000276:immunology*
chemical_list: D000906:Antibodies; D005456:Fluorescent Dyes; D014409:Tumor Necrosis Factor-alpha