國家衛生研究院 NHRI:Item 3990099045/5555
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    題名: Probing the binding kinetics of proinflammatory cytokine-antibody interactions using dual color fluorescence cross correlation spectroscopy
    作者: Wu, CY;Huang, CK;Chung, CY;Huang, IP;Hwu, Y;Yang, CS;Lai, YK;Lo, LW;Chiang, SY
    貢獻者: Division of Medical Engineering Research;Center for Nanomedicine Research
    摘要: 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.
    日期: 2011-03-29
    關聯: Analyst. 2011 Mar 29;136(10):2111-2118.
    Link to: http://dx.doi.org/10.1039/c0an00995d
    JIF/Ranking 2023: http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=NHRI&SrcApp=NHRI_IR&KeyISSN=0003-2654&DestApp=IC2JCR
    Cited Times(WOS): https://www.webofscience.com/wos/woscc/full-record/WOS:000289896000015
    Cited Times(Scopus): http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=79955141288
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