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Fluorescence correlation spectroscopy to study antibody binding and stoichiometry of complexes

机译：荧光相关光谱研究综合的抗体结合和化学计量

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FCS (fluorescence correlation spectroscopy) was used to study the association at the single molecule level of tumor necrosis factor alpha (TNF-α) and two of its protein antagonists Humira~(TM) (adalimumab), a fully humanized monoclonal antibody, and Enbrel~(TM) (etanercept), a soluble form of the TNF receptor. Single molecule approaches potentially have the advantage not only of enhanced sensitivity, but also of observing at equilibrium the details that would otherwise be lost in classical ensemble experiments where heterogeneity is averaged. We prepared fluorescent conjugates of the protein drugs and their biological target, the trimeric soluble form of TNF-α.^sThe bivalency of adalimumab and the trimeric nature of TNF-α potentially allow several forms of associative complexes that may differ in stoichiometry. Detailed knowledge of this reaction may be relevant to understanding adalimumab's pharmacological properties. Our FCS data showed that a single trimeric TNF-α can bind up to three adalimumab molecules. Under some conditions even larger complexes are formed, apparently the result of cross-linking of TNF-α trimers by adalimumab. In addition, distinct differences between Humira and Enbrel were observed in their association with TNF-α.
机译：FCS（荧光相关光谱）用于研究肿瘤坏死因子α（TNF-α）的单一分子水平的关联，其两种蛋白质拮抗剂Humira〜（Adalimalab），一种完全人源化单克隆抗体和嵌入式〜（TM）（依赖替辛），一种可溶形式的TNF受体。单个分子方法可能不仅具有增强的灵敏度，而且在均衡的细节下观察的优点是在经典集合实验中丢失的细节。我们制备了蛋白质药物的荧光缀合物及其生物靶标，TNF-α的三聚体可溶性形式。^ STHE ADALIMIMAB的二甲基苯和TNF-α的三聚体性质可能允许几种形式的化学计量不同的缔合复合物。对该反应的详细知识可能与了解Adalimalab的药理学特性有关。我们的FCS数据显示单个三聚体TNF-α可以结合到三个催风机组分子。在某些条件下，形成较大的复合物，显然是Adalimalab的TNF-α三聚体交联的结果。此外，在与TNF-α的关系中观察到肱杜拉和嵌入的不同差异。

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