High throughput solution-based measurement of antibody-antigen affinity and epitope binning.
High throughput solution-based measurement of antibody-antigen affinity and epitope binning.
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作者：
Estep P;Reid F;Nauman C;Liu Y;Sun T;Sun J;Xu Y
人类抗体发现的进步使人们能够选择数百种针对许多治疗相关靶点的高亲和力抗体。这就需要开发可重复的、高通量的分析技术来表征这些选择的输出。在这些特征中，表位覆盖率和亲和力是铅识别最关键的属性之一。Biolayer干涉术(BLI)由于其快速和低抗原消耗而成为一种有吸引力的表位结合技术。虽然基于表面的方法，如BLI和表面等离子体共振(SPR)通常用于亲和力测定，但传感器化学和表面相关伪影会限制高亲和力测量的准确性。当比较BLI和基于溶液平衡的动力学排除分析时，观察到测量的亲和力有显著差异(10倍以上)。KinExA直接缔合(Ka)速率常数的测量表明，这主要是由与BLI相关的表面现象相关的不准确的ka测量引起的。基于KinExA的动力学排斥分析原理，我们开发了一种高通量的96孔板格式分析，使用介观尺度发现(MSD)仪器来测量溶液的平衡亲和力。这种改进的方法结合了基于解的方法的准确性和以前只有基于表面的方法才能达到的吞吐量。
Advances in human antibody discovery have allowed for the selection of hundreds of high affinity antibodies against many therapeutically relevant targets. This has necessitated the development of reproducible, high throughput analytical techniques to characterize the output from these selections. Among these characterizations, epitopic coverage and affinity are among the most critical properties for lead identification. Biolayer interferometry (BLI) is an attractive technique for epitope binning due to its speed and low antigen consumption. While surface-based methods such as BLI and surface plasmon resonance (SPR) are commonly used for affinity determinations, sensor chemistry and surface related artifacts can limit the accuracy of high affinity measurements. When comparing BLI and solution equilibrium based kinetic exclusion assays, significant differences in measured affinity (10-fold and above) were observed. KinExA direct association (ka) rate constant measurements suggest that this is mainly caused by inaccurate ka measurements associated with BLI related surface phenomena. Based on the kinetic exclusion assay principle used for KinExA, we developed a high throughput 96-well plate format assay, using a Meso Scale Discovery (MSD) instrument, to measure solution equilibrium affinity. This improved method combines the accuracy of solution-based methods with the throughput formerly only achievable with surface-based methods.