State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, 510000, China.
Virol Sin. 2021 Jun;36(3):537-544. doi: 10.1007/s12250-020-00295-2. Epub 2020 Sep 29.
The assessment of neutralization activity is an important step in the evaluation of neutralizing antibodies (NAbs). The traditional methods for measuring the antibody neutralization of human adenovirus type 3 (HAdV-3) are the microneutralization (MN) assay, which has insufficient sensitivity, and the plaque reduction neutralization test (PRNT), which is not suitable for high-throughput screening. Herein, we describe the development of a flow cytometry-based neutralization (FCN) assay for measuring the neutralization of sera, cell culture supernatants, and chimeric antibodies against HAdV-3 on the basis of a recombinant HAdV-3 (rHAdV-3) construct expressing the enhanced green fluorescent protein (EGFP). For flow cytometry-based assays, the optimal cell confluence was determined as 90%, and the virus was titrated using the assay. The established FCN assay follows the percentage law and an optimal MOI of not less than 5 × 10 was determined by using a purified chimeric antibody. In addition, comparison of the anti-HAdV-3 NAb titers of 72 human serum samples by the MN and FCN assays, showed that both assays correlated strongly with each other. Our FCN assay was an improvement over the MN assay because the observation period was reduced from 3 to 1 days and data analysis could be performed objectively and robotically. Importantly, the newly established FCN assay allows measurement of the neutralization activity of chimeric antibodies expressed in cell culture supernatants. Thus, this sensitive and high-throughput FCN assay is a useful alternative to the MN assay for measuring the antibody neutralization of HAdV-3 and for screening anti-HAdV-3 NAbs in cell culture supernatants.
中和活性评估是评估中和抗体（NAb）的重要步骤。传统的测量人腺病毒 3 型（HAdV-3）抗体中和的方法是微量中和（MN）测定法，该方法灵敏度不足，而噬斑减少中和试验（PRNT）不适合高通量筛选。在此，我们描述了一种基于流式细胞术的中和（FCN）测定法的发展，该测定法基于表达增强型绿色荧光蛋白（EGFP）的重组 HAdV-3（rHAdV-3）构建体，用于测量针对 HAdV-3 的血清、细胞培养上清液和嵌合抗体的中和作用。对于基于流式细胞术的测定法，确定了最佳细胞汇合度为 90%，并使用该测定法滴定病毒。通过使用纯化的嵌合抗体，确定了建立的 FCN 测定法遵循百分比定律和最佳 MOI 不低于 5×10。此外，通过 MN 和 FCN 测定法比较 72 个人血清样本的抗 HAdV-3 NAb 效价，发现两种测定法均具有很强的相关性。我们的 FCN 测定法优于 MN 测定法，因为观察期从 3 天缩短至 1 天，并且可以客观地进行数据处理。重要的是，新建立的 FCN 测定法可以测量细胞培养上清液中表达的嵌合抗体的中和活性。因此，这种灵敏且高通量的 FCN 测定法是一种替代 MN 测定法的有用方法，可用于测量 HAdV-3 的抗体中和作用，并筛选细胞培养上清液中的抗 HAdV-3 NAb。
