Chen Peter, Mesci Aruz, Carlyle James R
Department of Immunology, University of Toronto and Sunnybrook Research Institute, Toronto, Ontario, Canada.
Methods Mol Biol. 2011;748:209-25. doi: 10.1007/978-1-61779-139-0_15.
Monoclonal antibodies (mAbs) specific for cell surface antigens are an invaluable tool to study immune receptor expression and function. Here, we outline a generalized reporter cell-based approach to the generation and high-throughput screening of mAbs specific for cell surface antigens. Termed CELLISA, this technology hinges upon the capture of hybridoma supernatants in mAb arrays that facilitate ligation of an antigen of interest displayed on BWZ reporter cells in the form of a CD3ζ-fusion chimeric antigen receptor (zCAR); in turn, specific mAb-mediated cross-linking of zCAR on BWZ cells results in the production of β-galactosidase enzyme (β-gal), which can be assayed colorimetrically. Importantly, the BWZ reporter cells bearing the zCAR of interest may be used for immunization as well as screening. In addition, serial immunizations employing additional zCAR- or native antigen-bearing cell lines can be used to increase the frequency of the desired antigen-specific hybridomas. Finally, the use of a cohort of epitope-tagged zCAR (e.g., zCAR(FLAG)) variants allows visualization of the cell surface antigen prior to immunization, and coimmunization using these variants can be used to enhance the immunogenicity of the target antigen. Employing the CELLISA strategy, we herein describe the generation of mAb directed against an uncharacterized natural killer cell receptor protein.
针对细胞表面抗原的单克隆抗体（mAb）是研究免疫受体表达和功能的宝贵工具。在此，我们概述了一种基于报告细胞的通用方法，用于生成和高通量筛选针对细胞表面抗原的单克隆抗体。这种技术称为CELLISA，它依赖于在单克隆抗体阵列中捕获杂交瘤上清液，该阵列有助于连接以CD3ζ融合嵌合抗原受体（zCAR）形式展示在BWZ报告细胞上的感兴趣抗原；反过来，zCAR在BWZ细胞上的特异性单克隆抗体介导的交联导致β-半乳糖苷酶（β-gal）的产生，可通过比色法进行检测。重要的是，携带感兴趣zCAR的BWZ报告细胞可用于免疫和筛选。此外，使用额外的携带zCAR或天然抗原的细胞系进行系列免疫可用于增加所需抗原特异性杂交瘤的频率。最后，使用一组表位标记的zCAR（例如，zCAR(FLAG)）变体可在免疫前可视化细胞表面抗原，并且使用这些变体进行共免疫可用于增强靶抗原的免疫原性。采用CELLISA策略，我们在此描述了针对一种未表征的自然杀伤细胞受体蛋白的单克隆抗体的产生。
