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Electrochemical measurement of antibody-antigen recognition biophysics: Thermodynamics and kinetics of human chorionic gonadotropin (hCG) binding to redox-tagged antibodies
Journal of Electroanalytical Chemistry ( IF 4.5 ) Pub Date : 2018-06-01 , DOI: 10.1016/j.jelechem.2018.02.062
Lee I. Partington , Stephen L. Atkin , Eric S. Kilpatrick , Samuel H. Morris , Mark Piper , Nathan S. Lawrence , Jay D. Wadhawan

Abstract The thermodynamics and kinetics of antigen binding under diffusive conditions to an electrode surface modified with ferrocene-tagged antibodies is studied within this work, and realised experimentally for the case of human chorionic gonadotropin (hGC) as the antigen with monoclonal anti-hCG antibodies immobilised on an electrode surface via a molecular wire platform. The formation of the antigen-antibody complex is monitored through the blocking of the ferrocene voltammetry, thereby enabling the fractional coverage of the electrode binding sites to be unravelled as a function of time. It is found that, at low antigen concentrations, a Frumkin adsorption isotherm fits the data, with repulsive interactions between bound antigens playing a significant role, with an affinity constant that is an order of magnitude larger than in the case of an untagged antibody, suggesting that the chemical hydrophobicity of the redox tag may encourage stronger binding. Comparison of the experimental temporal data with relevant diffusion-adsorption models under activation control allows for the extraction of the kinetic parameters; at zero coverage, the rate constants for adsorption and desorption are, respectively, larger and smaller than the untagged antibody. The kinetic study enables the confirmation that this type of platform may be utilised for rapid (15 min) and quantitative electroimmunoassay. This is validated through proof-of-concept analytical measurements, yielding a limit of detection around 25 mIU mL−1 (corresponding to 2.7 ng mL−1) – a value used clinically for urine hCG measurements corresponding to around four weeks of gestational age.

中文翻译:

抗体-抗原识别生物物理学的电化学测量:人绒毛膜促性腺激素 (hCG) 与氧化还原标记抗体结合的热力学和动力学

摘要 在这项工作中研究了在扩散条件下抗原与二茂铁标记抗体修饰的电极表面结合的热力学和动力学,并在以人绒毛膜促性腺激素 (hGC) 作为抗原的情况下通过实验实现,其中固定了单克隆抗 hCG 抗体。通过分子线平台在电极表面上。通过二茂铁伏安法的阻断来监测抗原-抗体复合物的形成,从而使电极结合位点的覆盖率随着时间的变化而被解开。发现,在低抗原浓度下,Frumkin 吸附等温线符合数据,结合抗原之间的排斥相互作用起着重要作用,亲和常数比未标记抗体的情况大一个数量级,表明氧化还原标签的化学疏水性可能会促进更强的结合。实验时间数据与激活控制下的相关扩散吸附模型的比较允许提取动力学参数;在零覆盖率下,吸附和解吸的速率常数分别大于和小于未标记抗体。动力学研究能够确认这种类型的平台可用于快速(15 分钟)和定量电免疫测定。这通过概念验证分析测量得到验证,产生大约 25 mIU mL-1(对应于 2.
更新日期:2018-06-01
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