Key Points A novel method to generate mAb using tetramers and reporter cells is described. Ab against the MCMV immunoevasin m12 are generated and characterized. An mAb was identified that is capable of blocking m12-mediated NK inhibition. Visual Abstract The generation of reliable mAb of unique and desired specificities serves as a valuable technology to study protein expression and function. However, standard approaches to mAb generation usually involve large-scale protein purification and intensive screening. In this study, we describe an optimized high-throughput proof-of-principle method for the expanded generation, enrichment, and screening of mouse hybridomas secreting mAb specific for a protein of interest. Briefly, we demonstrate that small amounts of a biotinylated protein of interest can be used to generate tetramers for use as prime-boost immunogens, followed by selective enrichment of Ag-specific B cells by magnetic sorting using the same tetramers prior to hybridoma generation. This serves two purposes: 1) to effectively expand both low- and high-affinity B cells specific for the antigenic bait during immunization and 2) to minimize subsequent laborious hybridoma efforts by positive selection of Ag-specific, Ab-secreting cells prior to hybridoma fusion and validation screening. Finally, we employ a rapid and inexpensive screening technology, CELLISA, a high-throughput validation method that uses a chimeric Ag fused to the CD3ζ signaling domain expressed on enzyme-generating reporter cells; these reporters can detect specific mAb in hybridoma supernatants via plate-bound Ab-capture arrays, thereby easing screening. Using this strategy, we generated and characterized novel mouse mAb specific for a viral immunoevasin, the mouse CMV m12 protein, and suggest that these mAb may protect mice from CMV infection via passive immunity.

中文翻译：

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四聚体免疫和选择，然后是 CELLISA 筛选以生成针对小鼠巨细胞病毒 m12 Immunoevasin 的单克隆抗体

要点 描述了一种使用四聚体和报告细胞生成 mAb 的新方法。产生并表征了针对 MCMV 免疫保护素 m12 的抗体。鉴定出一种能够阻断 m12 介导的 NK 抑制的 mAb。视觉摘要 生成具有独特和所需特异性的可靠 mAb 是研究蛋白质表达和功能的一项有价值的技术。然而，mAb 生成的标准方法通常涉及大规模蛋白质纯化和密集筛选。在这项研究中，我们描述了一种优化的高通量原理验证方法，用于扩展生成、富集和筛选分泌对感兴趣的蛋白质具有特异性的 mAb 的小鼠杂交瘤。简要地，我们证明了少量的感兴趣的生物素化蛋白质可用于生成四聚体，用作prime-boost 免疫原，然后在杂交瘤生成之前使用相同的四聚体通过磁性分选选择性富集 Ag 特异性 B 细胞。这有两个目的：1) 在免疫过程中有效扩增对抗原诱饵特异的低亲和力和高亲和力 B 细胞，2) 通过在杂交瘤之前阳性选择 Ag 特异性、Ab 分泌细胞，最大限度地减少随后繁琐的杂交瘤工作融合和验证筛选。最后，我们采用了一种快速且廉价的筛选技术 CELLISA，这是一种高通量验证方法，它使用与酶生成报告细胞上表达的 CD3ζ 信号结构域融合的嵌合 Ag；这些报告基因可以通过平板结合的抗体捕获阵列检测杂交瘤上清液中的特定 mAb，从而简化筛选。使用这种策略，我们生成并表征了病毒免疫逃逸蛋白（小鼠 CMV m12 蛋白）特异性的新型小鼠 mAb，并表明这些 mAb 可以通过被动免疫保护小鼠免受 CMV 感染。