PO.PR01.02 · 预防研究
用于快速检测HPV阳性口咽癌的HPV血清学
HPV serology for rapid detection of HPV positive oropharyngeal cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：HPV16（人乳头瘤病毒）是美国口咽癌（OPC）的主要病因。HPV16早期抗原抗体在诊断前长达二十年即可在血清样本中检测到，是早期检测的潜在生物标志物。在本研究中，我们开发并比较了三种用于检测OPC的定制HPV16血清学平台：MSD（Meso Scale Discovery）电化学发光检测、RAPID ELISA以及一种使用定制安卓应用程序的新型半定量侧向流检测（LFA）。
方法：对于MSD平台，重组HPV16抗原（E1、E2、E6、E7）在哺乳动物细胞中合成并印制在多重阵列上。在RAPID ELISA中，使用哺乳动物体外转录和翻译表达GST标签抗原（E1、E2、E6、E7），并检测结合的IgG。对于LFA，合成、印制His标签重组蛋白（HPV16 CE2 184-365aa和HPV16 E7），并用偶联蛋白G的纳米颗粒检测抗体。从新诊断的OPC患者中抽取的存档血清样本（n = 60）及配对唾液样本（n = 54，仅MSD检测）获自梅奥诊所。非配对对照（n = 46）获自AT Still。对于侧向流检测，使用已知浓度单克隆抗体的滴定曲线进行比色图像分析。我们通过多参数分析并绘制检测性能的受试者工作特征曲线优化了生物标志物组合（MSD和ELISA采用E2、E6和E7，LFA采用E2和E7），并使用Cohen's kappa统计量比较各检测方法。
结果：在98%特异性下，MSD HPV16血清学检测达到88%敏感性，与RAPID ELISA相似，LFA检测为85%敏感性。唾液样本的信号强度较弱，敏感性为24%。三种检测方法之间比较的Cohen's kappa值范围为0.74至0.78。在所有病例中，只有一个样本经三种血清学检测均为阴性。
结论：三种检测方法在HPV16早期抗原血清学检测方面具有很强的一致性。所有检测方法均可促进大规模HPV血清学研究。
查看英文原文 English abstract
Background: HPV16 (human papillomavirus) is a leading cause of oropharyngeal cancer (OPC) in the US. Antibodies to HPV16 early antigens are detectable in serum samples up to two decades before diagnosis and are potential biomarkers for early detection. In this study, we have developed and compared three custom HPV16 serology platforms for detection of OPC: MSD (Meso Scale Discovery) electrochemiluminescent detection, RAPID ELISA, and a novel semi-quantitative lateral flow assay (LFA) using a custom android application.
Methods: For the MSD platform, recombinant HPV 16 antigens (E1, E2, E6, E7) were synthesized in mammalian cells and printed on multiplexed arrays. In RAPID ELISA, GST-tagged antigens (E1, E2, E6, E7) were expressed using mammalian in vitro transcription and translation and bound IgG detected. For the LFA, His-tagged recombinant proteins (HPV16 CE2 184-365aa and HPV16 E7) were synthesized, printed, and antibodies detected with protein G conjugated nanoparticles. Archived serum samples drawn from newly-diagnosed OPC patients (n = 60), with matching saliva samples (n=54, MSD assay only) were obtained from Mayo Clinic. Unmatched controls (n = 46) were obtained from AT Still. For the lateral flow assay, colorimetric image analysis was done using a titration curve of known concentration of monoclonal antibody. We optimized the biomarker panel (E2, E6, and E7 for MSD and ELISA, and for LFA E2, and E7) by using multiparametric analysis and plotting the receiver operating curves to the test performance, and assays were compared using the Cohen's kappa statistic.
Results: At 98% specificity, the MSD HPV16 serologic assay achieved 88% sensitivity, similar to RAPID ELISA, and the LFA assay at 85% sensitivity. The signal intensities for saliva samples were weaker at 24% sensitivity. The Cohen's kappa value ranged from 0.74 to 0.78 for the comparison between all three assays. There was only one sample among all the cases that was negative by all three serology tests.
Conclusion: All three assays have strong concordance for the detection of HPV16 early antigen serology. All the tests can facilitate large scale HPV serology research.
利益披露 Disclosure
A. Kaushik, None..
D. Routman, None..
T. Patel, None..
J. Eger, None..
P. Yuvaraj, None..
K. R. Bartemes, None..
M. D. Griesel, None..
D. E. Hunter, None..
K. V. Abel, None.
A. Mathew,
Meso Scale Discovery Employment.
M. Wang,
Meso Scale Discovery Employment.
L. Dzantiev,
Meso Scale Discovery Employment.
M. Stengelin,
Meso Scale Discovery Employment.
J. N. Wohlstadter,
Meso Scale Discovery Employment.
A. Spolarich, None..
K. R. Dahlstrom, None.
J. B. Christen,
Flex biotech Stock Option.
E. Sturgis, None.
K. Anderson,
Flex Biotech Stock Option.