Department of Radiology and Biomedical Imaging, Yale University.
Department of Radiology and Biomedical Imaging, Yale University;
J Vis Exp. 2022 Jun 23(184). doi: 10.3791/63927.
Determining binding affinity (KD) is an important aspect of the characterization of radiolabeled antibodies (rAb). Typically, binding affinity is represented by the equilibrium dissociation constant, KD, and can be calculated as the concentration of antibody at which half the antibody binding sites are occupied at equilibrium. This method can be generalized to any radiolabeled antibody or other protein and peptide scaffolds. In contrast to cell-based methods, the choice of immobilized antigens is particularly useful for validating binding affinities after long-term storage of antibodies, distinguishing binding affinities of fragment antigen-binding region (Fab) arms in bispecific antibody constructs, and determining if there is variability in antigen expression between different cell lines. This method involves immobilizing a fixed amount of antigen to specified wells on a breakable 96-well plate. Then, nonspecific binding was blocked in all wells with bovine serum albumin (BSA). Subsequently, the rAb was added in a concentration gradient to all wells. A range of concentrations was chosen to allow the rAb to reach saturation, i.e., a concentration of antibody at which all antigens are continuously bound by the rAb. In designated wells without immobilized antigen, nonspecific binding of the rAb can be determined. By subtracting nonspecific binding from total binding in the wells with immobilized antigen, specific binding of the rAb to the antigen can be determined. The KD of the rAb was calculated from the resulting saturation binding curve. As an example, binding affinity was determined using radiolabeled amivantamab, a bispecific antibody for epidermal growth factor receptor (EGFR) and cytoplasmic mesenchymal-epithelial transition (cMET) proteins.
确定结合亲和力（KD）是放射性标记抗体（rAb）特性分析的一个重要方面。通常，结合亲和力由平衡解离常数 KD 表示，可以计算为抗体的浓度，在此浓度下，一半的抗体结合位点在平衡时被占据。这种方法可以推广到任何放射性标记的抗体或其他蛋白质和肽支架。与基于细胞的方法相比，固定化抗原的选择特别有用，可用于在抗体长期储存后验证结合亲和力，区分双特异性抗体构建体中片段抗原结合区（Fab）臂的结合亲和力，并确定不同细胞系之间抗原表达是否存在变异性。该方法涉及将固定量的抗原固定在可裂解 96 孔板的指定孔中。然后，在所有孔中用牛血清白蛋白（BSA）阻断非特异性结合。随后，将 rAb 以浓度梯度添加到所有孔中。选择一系列浓度以允许 rAb 达到饱和，即抗体的浓度，在该浓度下，所有抗原都被 rAb 连续结合。在没有固定化抗原的指定孔中，可以确定 rAb 的非特异性结合。通过从固定化抗原孔中的总结合中减去非特异性结合，可以确定 rAb 与抗原的特异性结合。从得到的饱和结合曲线计算 rAb 的 KD。例如，使用放射性标记的 amivantamab（一种针对表皮生长因子受体（EGFR）和细胞质间质上皮转化（cMET）蛋白的双特异性抗体）来确定结合亲和力。
