Department of Molecular and Medical Pharmacology, Crump Institute of Molecular Imaging, California NanoSystems Institute, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
Anal Biochem. 2010 Jun 15;401(2):173-81. doi: 10.1016/j.ab.2010.02.015. Epub 2010 Feb 21.
Many biological and biomedical laboratory assays require the use of antibodies and antibody fragments that strongly bind to their cell surface targets. Conventional binding assays, such as the enzyme-linked immunosorbent assay (ELISA) and flow cytometry, have many challenges, including capital equipment requirements, labor intensiveness, and large reagent and sample consumption. Although these techniques are successful in mainstream biology, there is an unmet need for a tool to quickly ascertain the relative binding capabilities of antibodies/antibody fragments to cell surface targets on the benchtop at low cost. We describe a novel cell capture assay that enables several candidate antibodies to be evaluated quickly as to their relative binding efficacies to their cell surface targets. We used chimeric rituximab and murine anti-CD20 monoclonal antibodies as cell capture agents on a functionalized microscope slide surface to assess their relative binding affinities based on how well they capture CD20-expressing mammalian cells. We found that these antibodies' concentration-dependent cell capture profiles correlate with their relative binding affinities. A key observation of this assay involved understanding how differences in capture surfaces affect the assay results. This approach can find utility when an antibody or antibody fragment against a known cell line needs to be selected for targeting studies.
许多生物和生物医学实验室检测都需要使用能够与细胞表面靶标强烈结合的抗体和抗体片段。传统的结合检测方法，如酶联免疫吸附测定（ELISA）和流式细胞术，存在许多挑战，包括资本设备要求、劳动强度大和试剂和样品消耗量大等问题。尽管这些技术在主流生物学中取得了成功，但仍然需要一种工具来快速确定抗体/抗体片段在低成本下对细胞表面靶标的相对结合能力。我们描述了一种新颖的细胞捕获检测方法，可快速评估几种候选抗体与其细胞表面靶标的相对结合效力。我们使用嵌合利妥昔单抗和鼠抗 CD20 单克隆抗体作为功能化显微镜载玻片表面上的细胞捕获剂，根据它们捕获表达 CD20 的哺乳动物细胞的能力来评估其相对结合亲和力。我们发现这些抗体的浓度依赖性细胞捕获曲线与它们的相对结合亲和力相关。该检测的一个关键观察结果涉及了解捕获表面的差异如何影响检测结果。当需要针对已知细胞系选择针对某个靶标的抗体或抗体片段时，这种方法可能具有实际应用价值。
