DEVELOPMENT/STANDARDIZATION/PRODUCT APPLICATION OF RETROVIRUS DETECTION ASSAYS

我们已经将努力方向转向开发用于以下的策略和测定： 在细胞基质中检测到低水平的逆转录病毒， 用于产品开发或异种移植的生物材料。我们 开发并标准化了一种改进的逆转录酶（RT） 用于检测需要Mg++的逆转录病毒（包括HIV，SIV， 和D型逆转录病毒。改进的RT分析可用于 生物制品中已知和新型逆转录病毒的检测 使用灵长类动物细胞基质。我们也在优化和规范 用于检测可能污染的其他逆转录病毒的RT试验 生物制品此外，我们还开发了灵敏的检测方法， 猴泡沫病毒（SFV）是一种潜在的污染物， 猿源性生物制品，包括疫苗和异种移植。这些 包括鉴定Mus dunni细胞系为高度易感 用于病毒分离;开发PCR引物，用于直接分析 感染的细胞和组织以及用于检测SFV的Western Blot和IFA 在猿和人类感染中的抗体。 细胞基质和生物材料及产品的评价 病毒污染对于产品安全是至关重要的。逆转录病毒序列 存在于所有物种中;大多数是有缺陷的，但在某些情况下， 感染性病毒的出现可能作为宿主的一个组成部分而持续存在 基因组，并可能导致疾病。因此，关键是 证明预期用于以下用途的生物材料中不存在逆转录病毒 人类使用。这些包括细胞基质，原材料，最终产品， 以及用于异种移植的组织/器官。因此，测定 检测低水平逆转录病毒污染以及检测 已知的以及新的逆转录病毒需要开发/优化， 产品安全性评价标准化。此外，经验 与分析也将使我们能够提供专家指导和审查 提交给食品药品管理局进行评估

We have directed efforts towards development of strategies and assays for the detection of low levels of retrovirus in cell substrates and biological materials used in product development or in xenotransplants. We have developed and standardized an improved reverse transcriptase (RT) assay for the detection of Mg++-requiring retroviruses including HIV, SIV, and type D retroviruses. The improved RT assay can be useful for the detection of known as well as novel retroviruses in biological products using a primate cell substrate. We are also optimizing and standardizing RT assays for the detection of other retroviruses which may contaminate biologics. In addition, we have developed sensitive detection assays for simian foamy virus (SFV) which is a potential contaminant of simian-derived biologics including vaccines and xenotransplants. These include identification of the Mus dunni cell line as highly susceptible for virus isolation; development of PCR primers for direct analysis of infected cells and tissues and Western Blot and IFA for detection of SFV antibodies in simian and human infections. Evaluation of cell substrates and biological materials and products for viral contamination is critical for product safety. Retroviral sequences are present in all species; the majority are defective but in some cases infectious viruses occur which may persist as an integrat part of the host genome and perhaps cause disease. Therefore, it is critical to demonstrate absence of retroviruses in biological materials intended for human use. These include cell substrates, raw materials, final products as well as tissues/organs used for xenotransplants. Thus, assays for detection of low level retrovirus contamination as well as for detection of known as well as novel retroviruses need to be developed/optimized and standardized for evaluation of product safety. Furthermore, experience with the assays will also enable us to provide expert guidance and review of submissions for evaluation at the FDA.