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Abstract：
The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (Kd), can be considered as one of the most important characteristics for any antibody-antigen pair. Many methods based on different technologies have been proposed and used to determine this value. However, since a very large number of publications and commercial datasheets do not include this information, significant obstacles in performing such measurements seem to exist. In other cases where such data are reported, the results have often proved to be unreliable. This situation may indicate that most of the technologies available today require a high level of expertise and effort that does not seem to be available in many laboratories. In this paper, we present a simple approach based on standard immunoassay technology that is easy and quick to perform. It relies on the effect that the molar IC50 approaches the Kd value in the case of infinitely small concentrations of the reagent concentrations. A two-dimensional dilution of the reagents leads to an asymptotic convergence to Kd. The approach has some similarity to the well-known checkerboard titration used for the optimization of immunoassays. A well-known antibody against the FLAG peptide, clone M2, was used as a model system and the results were compared with other methods. This approach could be used in any case where a competitive assay is available or can be developed. The determination of an affinity constant should belong to the crucial parameters in any quality control of antibody-related products and assays and should be mandatory in papers using immunochemical protocols.
摘要：
亲和力常数,也称为平衡常数，结合常数,平衡缔合常数，或倒数值，平衡解离常数(Kd)，可以被认为是任何抗体-抗原对的最重要特征之一。已经提出了许多基于不同技术的方法并用于确定该值。然而,由于大量出版物和商业数据表不包括这些信息，执行此类测量的重大障碍似乎存在。在报告此类数据的其他情况下，结果往往被证明是不可靠的。这种情况可能表明，当今可用的大多数技术都需要高水平的专业知识和努力，而许多实验室似乎都没有。在本文中,我们提出了一种基于标准免疫测定技术的简单方法，该方法易于快速执行。它依赖于在试剂浓度无限小浓度的情况下摩尔IC50接近Kd值的效果。试剂的二维稀释导致对Kd的渐近收敛。该方法与用于优化免疫测定的众所周知的棋盘滴定具有一些相似性。一种众所周知的抗FLAG肽的抗体，克隆M2作为模型系统，并将结果与其他方法进行比较。这种方法可以用于竞争性测定可用或可以开发的任何情况。亲和常数的确定应属于抗体相关产品和测定的任何质量控制中的关键参数，并且在使用免疫化学方案的论文中应该是强制性的。
