Patton Kathryn, Aslam Shahin, Lin Jim, Yu Li, Lambert Stacie, Dawes Glenn, Esser Mark T, Woo Jennifer, Janetzki Sylvia, Cherukuri Anu
Infectious Diseases/Vaccines Research, MedImmune, LLC, Mountain View, California, USA.
Clin Vaccine Immunol. 2014 May;21(5):628-35. doi: 10.1128/CVI.00736-13. Epub 2014 Feb 26.
Respiratory syncytial virus (RSV) causes significant disease in elderly adults, and we have previously reported that individuals 65 years of age and older have reduced RSV F protein-specific gamma interferon (IFN-γ)-producing T cells compared to healthy younger adults. To measure RSV F-specific memory T cell responses in the elderly following infection or vaccination, we optimized and qualified an IFN-γ enzyme-linked immunospot (ELISPOT) assay. Since peripheral blood mononuclear cells (PBMC) from the elderly could be more fragile, we established optimal cryopreservation techniques and minimal viability acceptance criteria. The number of cells per well, types and concentrations of stimulation antigens, and incubation times were evaluated to maximize assay sensitivity and precision. The optimized assay uses 300,000 cells/well, 2 μg/ml of an RSV F peptide pool (RSV Fpp), and incubation for 22 ± 2 h in serum-free CTL-Test medium. The assay was qualified by 3 analysts using 3 RSV F-responding donor PBMC samples (high, medium, and low responders) tested on 5 different assay days. The assay sensitivity or limit of detection (LOD) was determined to be 21 spot-forming cells (SFC) per 10(6) PBMC, and the lower limit of quantitation (LLOQ) was estimated to be 63 SFC/10(6) PBMC. The intra- and interassay percent coefficients of variation (CV) were <10.5% and <31%, respectively. The results of the qualification study demonstrate that a robust, precise, and sensitive IFN-γ ELISPOT assay has been developed that is fit for measuring RSV F-specific IFN-γ T cell responses in subjects enrolled in a vaccine clinical trial or in epidemiology studies.
呼吸道合胞病毒（RSV）可导致老年人患上严重疾病，我们之前曾报道，65岁及以上的个体与健康的年轻人相比，产生呼吸道合胞病毒F蛋白特异性γ干扰素（IFN-γ）的T细胞数量减少。为了测量老年人在感染或接种疫苗后呼吸道合胞病毒F特异性记忆T细胞反应，我们优化并验证了一种IFN-γ酶联免疫斑点（ELISPOT）检测方法。由于老年人的外周血单个核细胞（PBMC）可能更脆弱，我们建立了最佳冷冻保存技术和最低活力接受标准。对每孔细胞数量、刺激抗原的类型和浓度以及孵育时间进行了评估，以最大限度地提高检测的灵敏度和精密度。优化后的检测方法使用每孔300,000个细胞、2μg/ml的呼吸道合胞病毒F肽池（RSV Fpp），并在无血清CTL-Test培养基中孵育22±2小时。3名分析人员使用3份对呼吸道合胞病毒F有反应的供体PBMC样本（高、中、低反应者）在5个不同的检测日进行检测，对该检测方法进行了验证。检测灵敏度或检测限（LOD）确定为每10(6)个PBMC中有21个斑点形成细胞（SFC），定量下限（LLOQ）估计为63 SFC/10(6)个PBMC。批内和批间变异系数（CV）百分比分别<10.5%和<31%。验证研究结果表明，已开发出一种可靠、精确且灵敏的IFN-γ ELISPOT检测方法，适用于测量参与疫苗临床试验或流行病学研究的受试者中呼吸道合胞病毒F特异性IFN-γ T细胞反应。
