Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2
Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2
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DOI：
10.1038/sj.gt.3300946
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发表时间：
1999-07-01
期刊：
影响因子：
5.1
通讯作者：
Kleinschmidt, JA
中科院分区：
文献类型：
--
作者：
Grimm, D;Kern, A;Kleinschmidt, JA
我们证明了通过基于单克隆抗体的新型 ELISA 可以快速、可靠地定量物理 AAV-2（2 型腺相关病毒）颗粒，该单克隆抗体选择性识别组装的 AAV-2 衣壳。对多种重组 AAV-2 (rAAV) 制剂的滴定显示，至少 80% 的 aff 颗粒是空的，而野生型 AAV-2 储液中最多 50% 是空的，这表明重组基因组的衣壳化效率较低。这一发现在重组和野生型 AAV-2 母液的 CsCl 梯度组分滴定后得到证实。基于 ELISA 的衣壳数量测量显示，在组合中存在大量低密度物理颗粒，对应于空衣壳，但在野生型 AAV-2 制剂中却没有。此外，发现rAAV生产过程中VP蛋白的额外表达会导致衣壳过度形成，同时仅产生含DNA或转导rAAV颗粒的少量增加。我们的结论是，病毒基因组的衣壳化而不是衣壳组装可以限制 fbr rAAV 的产生，前提是维持 VP 表达的临界水平。通过 ELISA 定量 AAV-2 衣壳数量的可行性允许确定物理颗粒与含有 DNA 或感染性颗粒的比例。这些是重要的参数，应有助于优化和标准化重组 AAV-2 的生产和应用。
We demonstrate the rapid and reliable quantification of physical AAV-2 (adeno-associated virus type 2) particles via a novel ELISA based on a monoclonal antibody which selectively recognizes assembled AAV-2 capsids. Titration of a variety of recombinant AAV-2 (rAAV) preparations revealed that at least 80% of aff particles were empty, compared with a maximum of 50% in wild-type AAV-2 stocks, indicating that the recombinant genomes were less efficiently encapsidated. This finding was confirmed upon titration of CsCl gradient fractions from recombinant and wild-type AAV-2 stocks. ELISA-based measurement of capsid numbers revealed large number of physical particles with low densities corresponding to empty capsids in the combinant, but not in the wild-type AAV-2 preparations. Moreover, additional expression of VP proteins during rAAV production was found to result in an excessive capsid formation, whilst yielding only minor increases in DNA-containing or transducing rAAV particles. We conclude that encapsidation of viral genomes rather than capsid assembly can be limiting fbr rAAV production, provided that a critical level of VP expression is maintained The feasibility of quantifying AAV-2 capsid numbers via ther ELISA allows determination of physical to DNA-containing or infectious particle ratios These are important parameters which should help to optimize and standardize the production and application of recombinant AAV-2.