Optimization and validation of a multiplexed Luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses 6, 11, 16, and 18
Optimization and validation of a multiplexed Luminex assay to quantify antibodies to neutralizing epitopes on human papillomaviruses 6, 11, 16, and 18
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DOI：
10.1128/cdli.12.8.959-969.2005
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发表时间：
2005-08-01
期刊：
影响因子：
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通讯作者：
Esser, MT
中科院分区：
文献类型：
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作者：
Dias, D;Van Doren, J;Esser, MT
人乳头瘤病毒（HPV）多重竞争性Luminex免疫测定首先由Opalka等人描述（D.奥帕尔卡角E. Lachman，S. A. MacMullen，K.联合詹森，J.F.史密斯，N。Chirmule和AT. Esser，临床诊断实验室。10：108-115，2003）进行了优化和验证，以用于流行病学研究和疫苗临床试验。优化提高了分析灵敏度和临床特异性的测定，以更有效地区分HPV感染者的低滴度抗体反应从未感染的个人。优化的试验特征包括单克隆抗体（Nub）特异性、病毒样颗粒（VLP）与微球的结合比例放大、VLP浓度、Nub浓度、样品基质、样品稀释度、孵育时间、样品血清的热灭活和去污剂对试验缓冲液的影响。通过使用TECAN Genesis工作站使测定自动化，从而提高测定通量、再现性和操作员安全性。优化后，使用来自确定为HPV感染低风险和高风险个体的几种不同血清组对测定进行验证。然后使用经验证的测定法确定临床血清状态临界值。这种高通量检测方法已被证明可用于进行流行病学研究和评估预防性HPV疫苗的有效性。
A human papillomavirus (HPV) multiplexed competitive Luminex immunoassay first described by Opalka et al. (D. Opalka, C. E. Lachman, S. A. MacMullen, K. U. Jansen, J. F. Smith, N. Chirmule, and A T. Esser, Clin. Diagn. Lab. Immunol. 10:108-115, 2003) was optimized and validated for use in epidemiology studies and vaccine clinical trials. Optimization increased both the analytical sensitivity and the clinical specificity of the assay to more effectively discriminate the low-titer antibody response of HPV-infected persons from noninfected individuals. The characteristics of the assay that were optimized included monoclonal antibody (Nub) specificity, scaling up the conjugation of virus-like particles (VLPs) to microspheres, VLP concentration, Nub concentration, sample matrix, sample dilution, incubation time, heat inactivation of sample sera, and detergent effects on assay buffer. The assay was automated by use of a TECAN Genesis Workstation, thus improving assay throughput, reproducibility, and operator safety. Following optimization, the assay was validated using several distinct serum panels from individuals determined to be at low and high risk for HPV infection. The validated assay was then used to determine the clinical serostatus cutoff. This high-throughput assay has proven useful for performing epidemiology studies and evaluating the efficacy of prophylactic HPV vaccines.