Mesci Aruz, Carlyle James R
Department of Immunology, University of Toronto, Sunnybrook Research Institute, 2075 Bayview Ave, Toronto, Ontario, Canada M4N 3M5.
J Immunol Methods. 2007 May 31;323(1):78-87. doi: 10.1016/j.jim.2007.02.007. Epub 2007 Mar 15.
The generation of monoclonal antibodies (mAb) of desired specificity to cell surface antigens can serve as a valuable tool to study protein expression and function. However, traditional approaches to mAb generation usually involve large-scale protein purification and intensive screening, and may not result in mAb specificities to the native protein of interest. We describe a simple, inexpensive, high-throughput method for the generation and screening of hybridomas secreting mAb specific for cell surface receptors. Intact reporter cells expressing a CD3zeta-fusion receptor of the protein of interest are plated in 96-well arrays of captured, plate-bound hybridoma supernatants. A mAb to the protein of interest generates a signal leading to reporter-cell expression of beta-galactosidase, and enzyme activity can be screened in a single day using a non-radioactive substrate. Importantly, a single cell line can be used for immunization, screening, semi-quantitative affinity comparisons, and subsequent screening for physiological ligand expression, if the protein of interest is a receptor. We describe an application of this approach to generate mAb specific for a protein of previously unknown expression and undocumented function.
生成对细胞表面抗原有所需特异性的单克隆抗体（mAb）可作为研究蛋白质表达和功能的宝贵工具。然而，传统的单克隆抗体制备方法通常涉及大规模蛋白质纯化和密集筛选，并且可能无法产生针对目标天然蛋白质的单克隆抗体特异性。我们描述了一种简单、廉价、高通量的方法，用于生成和筛选分泌针对细胞表面受体的单克隆抗体的杂交瘤。将表达目标蛋白质的CD3ζ融合受体的完整报告细胞接种到捕获的、平板结合的杂交瘤上清液的96孔阵列中。针对目标蛋白质的单克隆抗体产生信号，导致报告细胞表达β-半乳糖苷酶，并且可以使用非放射性底物在一天内筛选酶活性。重要的是，如果目标蛋白质是一种受体，单个细胞系可用于免疫、筛选、半定量亲和力比较以及随后筛选生理配体表达。我们描述了这种方法的一种应用，以生成针对先前未知表达和未记录功能的蛋白质的单克隆抗体。
