Label-free epitope binning assays of monoclonal antibodies enable the identification of antigen heterogeneity
Label-free epitope binning assays of monoclonal antibodies enable the identification of antigen heterogeneity
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DOI：
10.1016/j.jim.2012.05.010
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发表时间：
2012-08-31
影响因子：
2.2
通讯作者：
Pons, Jaume
中科院分区：
文献类型：
--
作者：
Abdiche, Yasmina N.;Lindquist, Kevin C.;Pons, Jaume
无标记生物传感器通常用于发现治疗性抗体以表征靶向特定抗原的一组单克隆抗体的表位结合区，从而促进其组织成表位组或“箱”。当以成对组合方式测试时，彼此竞争结合特定抗原的两种抗体可以被分组成相同的表位箱-即，它们识别相似或重叠的表位-而同时结合抗原的两种抗体被置于不同的表位箱中。然而，取决于所使用的测定形式，这些实验的结果有时可能相互矛盾。在这里，我们提供了两个例子，说明抗原异质性，无论是在抗原样品中固有的，或由测定条件诱导，可以混淆表位分箱结果的解释，并在某些情况下，导致错误的结论。我们强调了为什么采用溶液抗原的测定通常比采用固定化抗原的测定更可靠，并且通过利用天然抗原的测定来证实我们的分组结果，我们确定了我们的异质性抗原样品的哪些亚群是生物相关的，从而提高了表位箱和功能活性之间的相关性。此外，我们还提供了进行确定性分组检测的建议，以及在怀疑抗原异质性时可以遵循的诊断检测程序。(C)2012 Elsevier B. V.保留所有权利。
Label-free biosensors are often used in the discovery of therapeutic antibodies to characterize the epitope binding regions of a panel of monoclonal antibodies that target a specific antigen, thus facilitating their organization into epitope groups or "bins". When tested in a pairwise combinatorial manner, two antibodies that compete with one another for binding to a specific antigen may be grouped into the same epitope bin - that is, they recognize similar or overlapping epitopes - whereas two antibodies that bind simultaneously to the antigen are placed into different epitope bins. However, depending on the assay format used, results from such experiments can sometimes contradict one another. Here, we provide two examples that illustrate how antigen heterogeneity, either inherent in an antigen sample, or induced by the assay conditions, can confound the interpretation of epitope binning results and, in some cases, lead to erroneous conclusions. We highlight why assays that employ solution antigen are often more reliable than those that employ immobilized antigen and, by corroborating our binning results with assays that utilize native antigen, we determine which subpopulations of our heterogeneous antigen samples are biologically relevant and thus improve the correlation between epitope bins and functional activity. Furthermore, we provide recommendations for performing definitive binning assays and a diagnostic assay procedure that can be followed when antigen heterogeneity is suspected. (C) 2012 Elsevier B.V. All rights reserved.