Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, FDA, Bethesda, MA, USA.
Gene Ther. 2013 Jan;20(1):43-50. doi: 10.1038/gt.2011.212. Epub 2012 Jan 19.
The availability of rapid and quantitative titration assays for retroviral vectors is important, especially in the context of clinical applications. In this report, we describe a novel assay to titrate lentiviral and gamma retroviral vectors. This rapid assay is based on protein fragment complementation involving the N-terminal (Bla1) and the C-terminal (Bla2) fragments of TEM-1 β-lactamase (BLAK). The Bla1 protein fragment is incorporated in the vector's envelope during vector production. Bla1-bearing vectors are titrated on Bla2-expressing cells. Upon transduction, Bla1 and Bla2 heterodimerize and restore BLAK's enzymatic function. The enzymatic activity of BLAK is quantified by flow cytometry using the green fluorescent CCF2/AM substrate, which is converted into a blue fluorescent product. The enzymatic conversion of the CCF2/AM substrate was found to be directly related to vector entry, as a neutralizing antibody completely blocked the conversion. The titers obtained using this rapid assay correlated well with the titers measured by functional transduction assays. The whole assay can be finished within 8 h. Thus, it is considerably less time consuming compared with other transduction-based titration assays for lentiviral and gamma retroviral vectors.
快速定量滴定分析逆转录病毒载体非常重要，特别是在临床应用方面。在本报告中，我们描述了一种新型的测定慢病毒和γ逆转录病毒载体滴度的方法。该快速测定法基于涉及 TEM-1 内酰胺酶（BLAK）的 N 端（Bla1）和 C 端（Bla2）片段的蛋白片段互补。Bla1 蛋白片段在载体生产过程中被整合到载体的包膜中。Bla1 载体在表达 Bla2 的细胞上滴定。转导后，Bla1 和 Bla2 异源二聚化并恢复 BLAK 的酶活性。通过使用绿色荧光 CCF2/AM 底物的流式细胞术定量测定 BLAK 的酶活性，该底物被转化为蓝色荧光产物。酶促转化的 CCF2/AM 底物与载体进入直接相关，因为中和抗体完全阻断了转化。使用这种快速测定法获得的滴度与通过功能性转导测定法测量的滴度很好地相关。整个测定可在 8 小时内完成。因此，与其他用于慢病毒和γ逆转录病毒载体的基于转导的滴定测定法相比，该测定法的耗时明显更少。
