A Generic Method for Fast and Sensitive Detection of Adeno-Associated Viruses Using Modified AAV Receptor Recombinant Proteins
A Generic Method for Fast and Sensitive Detection of Adeno-Associated Viruses Using Modified AAV Receptor Recombinant Proteins
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DOI：
10.3390/molecules24213973
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发表时间：
2019-11-01
期刊：
影响因子：
4.6
通讯作者：
Ding, Wei
中科院分区：
文献类型：
--
作者：
Cui, Mengtian;Lu, Yabin;Ding, Wei
腺相关病毒（AAV）是临床和实验室研究中广泛使用的基因治疗载体。不同AAV制剂的滴定对于质量控制目的以及在比较研究中是重要的。然而，目前可用的方法在它们以灵敏度和方便性检测各种血清型的能力方面是有限的。在这里，我们利用了一个新发现的AAV受体蛋白与多种AAV血清型的高亲和力，并开发了ELISA样方法命名为“VIRTUISA”（病毒受体连接的免疫吸附试验），通过采用与链霉亲和素结合肽（SBP）融合。已证明优化的VISAISA测定法对于AAV2的滴定表现出令人满意的性能。AAV2的线性范围为1 × 10（5）v.g.至5 x 10（9）v.g.，LOD（检测限）为5 x 10（4）v.g.用于定量AAV1的VISAISA测试也是成功的。我们的研究表明，用于定量不同血清型的AAV的通用方案是可行的，可靠的和具有成本效益的。VIRTUISA的应用不仅由于其简单性而有益于实验室研究，而且还可能用于监测给定AAV血清型的临床试验和野生型感染中的循环AAV载量。
Adeno-Associated Viruses (AAV) are widely used gene-therapy vectors for both clinical applications and laboratory investigations. The titering of different AAV preparations is important for quality control purposes, as well as in comparative studies. However, currently available methods are limited in their ability to detect various serotypes with sensitivity and convenience. Here, we took advantage of a newly discovered AAV receptor protein with high affinity to multiple AAV serotypes, and developed an ELISA-like method named "VIRELISA" (virus receptor-linked immunosorbent assay) by adopting fusion with a streptavidin-binding peptide (SBP). It was demonstrated that optimized VIRELISA assays exhibited satisfactory performance for the titering of AAV2. The linear range of AAV2 was 1 x 10(5) v.g. to 5 x 10(9) v.g., with an LOD (limit of detection) of 5 x 10(4) v.g. Testing of VIRELISA for the quantification of AAV1 was also successful. Our study indicated that a generic protocol for the quantification of different serotypes of AAVs was feasible, reliable and cost-efficient. The applications of VIRELISA will not only be of benefit to laboratory research due to its simplicity, but could also potentially be used for monitoring the circulation AAV loads both in clinical trials and in wild type infection of a given AAV serotype.