Immunology Core, Malaria Biologics Branch, WRAIR, 503 Robert Grant Ave, 3W58, Silver Spring, MD, 20910, USA.
Biotechnology HPC Software Applications Institute, Telemedicine and Advanced Technology Research Center, U.S. Army Medical Research and Development Command, Fort Detrick, Silver Spring, MD, 21702, USA.
Malar J. 2020 Apr 17;19(1):159. doi: 10.1186/s12936-020-03225-5.
Profiling immune responses induced by either infection or vaccination can provide insight into identification of correlates of protection. Furthermore, profiling of serological responses can be used to identify biomarkers indicative of exposure to pathogens. Conducting such immune surveillance requires readout methods that are high-throughput, robust, and require small sample volumes. While the enzyme-linked immunosorbent assay (ELISA) is the classical readout method for assessing serological responses, the advent of multiplex assays has significantly increased the throughput and capacity for immunoprofiling. This report describes the development and assay performance (sensitivity, linearity of detection, requirement for multiple dilutions for each sample, intra- and inter-assay variability) of an electro-chemiluminescence (ECLIA)-based multiplex assay.
The current study describes the development of a multiplex ECLIA-based assay and characterizes the sensitivity, linear range, and inter- and intra-assay variability of the ECLIA platform and its agreement with the traditional ELISA. Special emphasis was placed on potential antigenic competition when testing closely related antigens in the multiplex format.
Multiplexing of antigens in ECLIA provides significant practical benefits in terms of reducing sample volume requirements and experimental time. Beyond the practical advantages of multiplexing, the ECLIA provides superior assay performance when compared to the ELISA. Not only does ECLIA show good agreement with the ELISA assay, but the linear range of ECLIA is also sufficiently wide to permit single-dilution measurements of concentration without the need to do serial dilutions. The lack of antigenic competition allows the simultaneous testing of closely related antigens, such as plate antigens representing different alleles of the same protein, which can inform about cross-reactivities-or lack thereof-of serological responses.
The advantages of the newly developed tool for assessing the antigen profiles of serological responses may ultimately lead to the identification of biomarkers associated with various disease stages and or protection against disease.
分析感染或接种疫苗引起的免疫反应可以深入了解保护相关因素。此外，对血清反应谱的分析可用于鉴定表明接触病原体的生物标志物。进行这种免疫监测需要使用高通量、稳健且需要小样本量的读出方法。虽然酶联免疫吸附测定（ELISA）是评估血清反应的经典读出方法，但多重测定的出现大大提高了免疫分析的通量和能力。本报告介绍了基于电化学发光（ECLIA）的多重测定的开发和测定性能（灵敏度、检测线性、每个样品所需的多个稀释倍数、内和间测定变异性）。
本研究描述了一种基于多重 ECLIA 的测定的开发，并描述了 ECLIA 平台的灵敏度、线性范围以及内和间测定变异性，以及其与传统 ELISA 的一致性。当以多重格式测试密切相关的抗原时，特别强调了潜在的抗原竞争。
在 ECLIA 中对抗原进行多重化在减少样品体积需求和实验时间方面具有显著的实际优势。除了多重化的实际优势外，ECLIA 与 ELISA 相比具有优越的测定性能。ECLIA 不仅与 ELISA 测定具有良好的一致性，而且 ECLIA 的线性范围也足够宽，可以允许在无需进行连续稀释的情况下，对浓度进行单一稀释测量。缺乏抗原竞争允许同时测试密切相关的抗原，例如代表同一蛋白质不同等位基因的平板抗原，这可以提供有关血清反应的交叉反应性或缺乏交叉反应性的信息。
用于评估血清反应抗原谱的新工具的优势最终可能导致确定与各种疾病阶段相关的生物标志物，或者确定对疾病的保护作用。
