Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.
Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.
J Immunol Methods. 2019 Jul;470:6-14. doi: 10.1016/j.jim.2019.04.003. Epub 2019 Apr 18.
Enterotoxigenic Escherichia coli (ETEC) is a leading cause of bacterial diarrhea both among children in low and middle income countries and in travelers to these regions. Although there are several approaches to develop an effective vaccine for ETEC, no licensed vaccines are currently available. The most advanced ETEC vaccine candidates include multiple colonization factors along with the heat labile toxin B subunit. In the absence of known correlates of protection, and to understand the mechanism of protection, monitoring immune responses to a majority of the vaccine associated antigens using various types of samples is needed. Unfortunately, a traditional ELISA is time consuming, labor intensive and requires substantial amounts of antigens and sample volumes. To address these constraints, we developed and validated a novel high throughput electrochemiluminescent (ECL) - based multiplex immunoassay using Meso Scale Discovery (MSD) platform for analyzing immune responses to ETEC antigens. The ETEC multiplex ECL assay is an 8-plex assay which includes the ETEC colonization factor antigens (CFA/I, CS1, CS2, CS3, CS5 and CS6) along with the two subunits of heat labile toxin (LTA and LTB). Our data suggested that a single dilution of sample provides a quantifiable result for a wide range of sample titers. To compare ETEC multiplex ECL with ELISA, we carried out assays using the same antigens with the two immunoassay platforms using a common sample set of serum and ALS (antibodies in lymphocyte supernatant) specimens. The MSD platform achieved excellent correlations with ELISA for the antigens tested, consistently detecting comparable antibody levels in the samples. The ETEC multiplex ECL can serve as a fundamental platform in evaluating performances of candidate ETEC vaccines in future field trials.
肠产毒性大肠杆菌（ETEC）是中低收入国家儿童和前往这些地区的旅行者细菌性腹泻的主要原因。尽管有几种方法可以开发有效的 ETEC 疫苗，但目前没有许可的疫苗。最先进的 ETEC 疫苗候选物包括多种定植因子以及不耐热毒素 B 亚单位。由于缺乏已知的保护相关性，为了了解保护机制，需要使用各种类型的样本监测针对大多数疫苗相关抗原的免疫反应。不幸的是，传统的 ELISA 耗时、劳动密集且需要大量的抗原和样本量。为了解决这些限制，我们使用 Meso Scale Discovery（MSD）平台开发并验证了一种新型高通量电化学发光（ECL）-基于多重免疫分析，用于分析针对 ETEC 抗原的免疫反应。ETEC 多重 ECL 分析是一种 8 重分析，包括 ETEC 定植因子抗原（CFA/I、CS1、CS2、CS3、CS5 和 CS6）以及不耐热毒素的两个亚单位（LTA 和 LTB）。我们的数据表明，样品的单一稀释度可为广泛的样品滴度提供可量化的结果。为了比较 ETEC 多重 ECL 与 ELISA，我们使用两种免疫分析平台使用相同的抗原和共同的血清和 ALS（淋巴细胞上清液中的抗体）标本进行了检测。MSD 平台与 ELISA 测试的抗原具有出色的相关性，始终能够在样本中检测到可比的抗体水平。ETEC 多重 ECL 可以作为评估未来现场试验中候选 ETEC 疫苗性能的基本平台。
