Mauthe Alexandra, Cedrone Edward, Villar-Hernández Raquel, Rusch Elisa, Springer Marco, Schuster Martin, Preyer Rosemarie, Dobrovolskaia Marina A, Gutekunst Matthias
Department Immune Analytics, Genome Identification Diagnostics GmbH, Strassberg, Germany.
Nanotechnology Characterization Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research Sponsored by the National Cancer Institute, Frederick, MD, United States of America.
AAPS J. 2025 Apr 25;27(4):81. doi: 10.1208/s12248-025-01072-3.
The enzyme-linked immunosorbent spot (EliSpot) assay and its fluorescence-based version, FluoroSpot, are sensitive immunoassays commonly used to quantify antigen-specific T and B lymphocytes and other immune cells in peripheral blood or homogenized tissues. Due to their high sensitivity, these assays are popular in clinical trials to evaluate the efficacy of immunotherapy and vaccines, which involve a high level of scrutiny to ensure valid study results. Besides industry consensus white papers and other research publications, there is no formal guidance for the industry on how to validate EliSpot and FluoroSpot assays to ensure their accurate performance for immune monitoring in clinical trials. Herein, we describe a comprehensive in vitro study using healthy human donor peripheral blood mononuclear cells (PBMCs) and model antigens to validate a double-color FluoroSpot assay for monitoring antigen-specific lymphocytes by detecting and quantifying IFN-γ and IL-2-producing lymphocytes. Validation parameters, acceptance criteria set-up, and assay limits-limit of detection (LOD), minimum positive control response, lower and upper limits of quantification (LLOQ and ULOQ)-were determined, and assay performance was demonstrated by assessing precision, specificity, linearity, and robustness. In addition, an inter-laboratory comparison demonstrated concordance between assay results from two laboratories. In summary, this study outlines a robust approach to EliSpot and FluoroSpot validation and demonstrates that the IFN-γ/IL-2 FluoroSpot assay is suitable for the reliable detection of antigen-specific immune responses from PBMC samples across laboratories and meets the current regulatory requirements for bioanalytical method validation.
酶联免疫斑点（EliSpot）测定法及其基于荧光的版本FluoroSpot是常用的灵敏免疫测定法，用于定量外周血或匀浆组织中的抗原特异性T和B淋巴细胞以及其他免疫细胞。由于其高灵敏度，这些测定法在评估免疫疗法和疫苗疗效的临床试验中很受欢迎，而这些试验需要高度严格的审查以确保获得有效的研究结果。除了行业共识白皮书和其他研究出版物外，目前尚无针对该行业的关于如何验证EliSpot和FluoroSpot测定法以确保其在临床试验中进行免疫监测时准确性能的正式指南。在此，我们描述了一项全面的体外研究，使用健康人类供体的外周血单核细胞（PBMC）和模型抗原来验证一种双色FluoroSpot测定法，通过检测和定量产生IFN-γ和IL-2的淋巴细胞来监测抗原特异性淋巴细胞。确定了验证参数、验收标准设置以及测定限——检测限（LOD）、最小阳性对照反应、定量下限和上限（LLOQ和ULOQ），并通过评估精密度、特异性、线性和稳健性来证明测定性能。此外，一项实验室间比较证明了两个实验室的测定结果具有一致性。总之，本研究概述了一种用于EliSpot和FluoroSpot验证的稳健方法，并证明IFN-γ/IL-2 FluoroSpot测定法适用于跨实验室可靠检测PBMC样品中的抗原特异性免疫反应，并且符合生物分析方法验证的当前监管要求。
