Santos Radleigh, Buying Alcinette, Sabri Nazila, Yu John, Gringeri Anthony, Bender James, Janetzki Sylvia, Pinilla Clemencia, Judkowski Valeria A
Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway Port St. Lucie, FL 34987, USA.
Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121, USA.
Cells. 2014 Dec 24;4(1):1-18. doi: 10.3390/cells4010001.
Immune monitoring of functional responses is a fundamental parameter to establish correlates of protection in clinical trials evaluating vaccines and therapies to boost antigen-specific responses. The IFNg ELISPOT assay is a well-standardized and validated method for the determination of functional IFNg-producing T-cells in peripheral blood mononuclear cells (PBMC); however, its performance greatly depends on the quality and integrity of the cryopreserved PBMC. Here, we investigate the effect of overnight (ON) resting of the PBMC on the detection of CD8-restricted peptide-specific responses by IFNg ELISPOT. The study used PBMC from healthy donors to evaluate the CD8 T-cell response to five pooled or individual HLA-A2 viral peptides. The results were analyzed using a modification of the existing distribution free resampling (DFR) recommended for the analysis of ELISPOT data to ensure the most rigorous possible standard of significance. The results of the study demonstrate that ON resting of PBMC samples prior to IFNg ELISPOT increases both the magnitude and the statistical significance of the responses. In addition, a comparison of the results with a 13-day preculture of PBMC with the peptides before testing demonstrates that ON resting is sufficient for the efficient evaluation of immune functioning.
对功能性反应进行免疫监测是在评估疫苗和疗法以增强抗原特异性反应的临床试验中确定保护相关性的一个基本参数。IFNg ELISPOT检测是一种用于测定外周血单核细胞（PBMC）中产生功能性IFNg的T细胞的标准化且经过验证的方法；然而，其性能很大程度上取决于冻存PBMC的质量和完整性。在此，我们研究PBMC过夜静置对通过IFNg ELISPOT检测CD8限制性肽特异性反应的影响。该研究使用健康供体的PBMC来评估CD8 T细胞对五种混合或单个HLA - A2病毒肽的反应。使用对现有用于ELISPOT数据分析的分布自由重采样（DFR）方法的一种改进来分析结果，以确保尽可能严格的显著性标准。研究结果表明，在进行IFNg ELISPOT检测之前对PBMC样本进行过夜静置会增加反应的幅度和统计学显著性。此外，将结果与在测试前对PBMC与肽进行13天预培养的结果进行比较表明，过夜静置足以有效评估免疫功能。
