恒河猴血清中预存抗AAV8衣壳总抗体检测方法的建立与确认  

Establishment and Confirmation of a Method for Detection of Total Anti-capsid of AAV8 Antibodies in Serum of Rhesus Monkeys

作　　者：霍一凡 徐嘉辰 黄美晴 王国凤 邱云良 HUO Yifan;XU Jiachen;HUANG Meiqing;WANG Guofeng;QIU Yunliang(China State Institute of Pharmaceutical Industry,Shanghai 201203;Shanghai InnoStar Bio-Tech Co.,Ltd.,Shanghai 201203)

机构地区：[1]中国医药工业研究总院,上海201203 [2]上海益诺思生物技术股份有限公司,上海201203

出　　处：《中国医药工业杂志》2024年第6期851-859,865,共10页Chinese Journal of Pharmaceuticals

基　　金：上海市科委工程技术研究中心专项(17DZ2252900)。

摘　　要：随着基于腺相关病毒(AAV)基因治疗的显著发展,一项重要的任务是开发简便、快速且可靠的检测AAV预存抗体的方法。该研究基于酶联免疫吸附试验(ELISA),将AAV8衣壳包被于96孔板,采用重组蛋白A/G为检测抗体,建立了一种简单、快速检测恒河猴血清中抗AAV8衣壳总抗体(TAb)的方法,并对其进行方法学验证。结果表明,该方法的乘法校正因子为1.08,确证临界值为29.5%;筛选和确证灵敏度分别为43.1、76.5 ng/mL。1000、100、50 ng/mL的阳性对照抗体可耐受AAV8滴度分别为1.25×1010、3.13×10~9、1.56×10~9 GC/mL。批内、批间变异系数(CV)≤20.0%。此外,本方法有足够的选择性和样本稳定性,可用于恒河猴血清中预存抗AAV8衣壳TAb的检测。With the development of gene therapy based on adeno-associated virus(AAV),an important task is to develop a simple,rapid and reliable method for detecting pre-existing anti-AAV antibodies.In this study,a simple and rapid method for the detection of total antibodies(TAb)against AAV8 capsid in serum of rhesus monkeys was developed and confirmed.This method based on enzyme-linked immunosorbent assay(ELISA),and AAV8 capsid was coated on 96-well plate,recombinant protein A/G was used as the detection antibody.The results showed that the multiplicative correction factor of this assay was 1.08 and confirmatory cut point was 29.5%.The screening and confirmatory sensitivities were 43.1 and 76.5 ng/mL,respectively.The positive controls of 1000,100,50 ng/mL were tolerant to 1.25×10',3.13×10°,1.56x10°GC/mL AAV8,respectively.Both the coeficients of variation(CV)of intra-run and inter-run met CV≤20.0%.In addition,the method has suficient selectivity and sample stability,which can be used for TAb detection of pre-existing anti-AAV8 capsid in serum of rhesus monkeys.

关 键 词：AAV8 免疫原性 预存抗体 总抗体 ELISA 

分 类 号：R917[医药卫生—药物分析学]