Nath Nidhi, Godat Becky, Benink Hélène, Urh Marjeta
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, United States.
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, United States.
J Immunol Methods. 2015 Nov;426:95-103. doi: 10.1016/j.jim.2015.08.008. Epub 2015 Aug 24.
Antibodies labeled with small molecules such as fluorophore, biotin or drugs play an important role in various areas of biological research, drug discovery and diagnostics. However, the majority of current methods for labeling antibodies is solution-based and has several limitations including the need for purified antibodies at high concentrations and multiple buffer exchange steps. In this study, a method (on-bead conjugation) is described that addresses these limitations by combining antibody purification and conjugation in a single workflow. This method uses high capacity-magnetic Protein A or Protein G beads to capture antibodies directly from cell media followed by conjugation with small molecules and elution of conjugated antibodies from the beads. High-capacity magnetic antibody capture beads are key to this method and were developed by combining porous and hydrophilic cellulose beads with oriented immobilization of Protein A and Protein G using HaloTag technology. With a variety of fluorophores it is shown that the on-bead conjugation method is compatible with both thiol- and amine-based chemistry. This method enables simple and rapid processing of multiple samples in parallel with high-efficiency antibody recovery. It is further shown that recovered antibodies are functional and compatible with downstream applications.
用荧光团、生物素或药物等小分子标记的抗体在生物学研究、药物发现和诊断等各个领域发挥着重要作用。然而，目前大多数抗体标记方法基于溶液，存在若干局限性，包括需要高浓度的纯化抗体以及多个缓冲液交换步骤。在本研究中，描述了一种方法（磁珠偶联法），该方法通过在单个工作流程中结合抗体纯化和偶联来解决这些局限性。此方法使用高容量的磁性蛋白A或蛋白G磁珠直接从细胞培养基中捕获抗体，随后与小分子偶联，并从磁珠上洗脱偶联的抗体。高容量磁性抗体捕获磁珠是该方法的关键，它是通过将多孔和亲水的纤维素磁珠与使用HaloTag技术定向固定的蛋白A和蛋白G相结合而开发的。使用多种荧光团表明，磁珠偶联法与基于硫醇和胺的化学方法均兼容。该方法能够简单快速地并行处理多个样品，并高效回收抗体。进一步表明，回收的抗体具有功能性且与下游应用兼容。
