Characterization of monoclonal antibody's binding kinetics using oblique-incidence reflectivity difference approach.
Characterization of monoclonal antibody's binding kinetics using oblique-incidence reflectivity difference approach.
复制标题
使用倾斜的反射率差异方法来表征单克隆抗体的结合动力学。
作者：
Liu S;Zhang H;Dai J;Hu S;Pino I;Eichinger DJ;Lyu H;Zhu H
抗人蛋白质的单克隆抗体（mAb）是基础研究、诊断和分子治疗学的主要蛋白质捕获试剂。在所有这些应用中使用的mAb的2个最重要的属性是它们的特异性和亲合力。虽然抗人蛋白质的mAb的特异性可以基于其在人蛋白质组微阵列上的结合谱容易地定义，但以高通量和成本有效的方式确定mAb的亲合力值一直是一个挑战。为了应对这一挑战，我们采用了入射反射率差（OIRD）平台来表征蛋白质微阵列格式中的mAb。我们首先用OIRD方法系统地测定了50个单克隆抗体的Kon和Koff值，并推导出亲合力值。其次，我们建立了一种多重方法，同时测量9种单特异性mAb的混合物的亲合力值，这些mAb不会与抗原发生交叉反应。第三，我们证明了可以使用流动池装置顺序确定一组mAb的亲合力值。最后，我们实施了顺序竞争测定，使我们能够结合识别相同抗原的多个mAb。我们的研究表明，OIRD提供了一个高通量和具有成本效益的平台，用于表征单克隆抗体的结合动力学。
Monoclonal antibodies (mAbs) against human proteins are the primary protein capture reagents for basic research, diagnosis, and molecular therapeutics. The 2 most important attributes of mAbs used in all of these applications are their specificity and avidity. While specificity of a mAb raised against a human protein can be readily defined based on its binding profile on a human proteome microarray, it has been a challenge to determine avidity values for mAbs in a high-throughput and cost-effective fashion. To undertake this challenge, we employed the oblique-incidence reflectivity difference (OIRD) platform to characterize mAbs in a protein microarray format. We first systematically determined the Kon and Koff values of 50 mAbs measured with the OIRD method and deduced the avidity values. Second, we established a multiplexed approach that simultaneously measured avidity values of a mixture of 9 mono-specific mAbs that do not cross-react to the antigens. Third, we demonstrated that avidity values of a group of mAbs could be sequentially determined using a flow-cell device. Finally, we implemented a sequential competition assay that allowed us to bin multiple mAbs that recognize the same antigens. Our study demonstrated that OIRD offers a high-throughput and cost-effective platform for characterization of the binding kinetics of mAbs.