Validation of a HLA-A2 tetramer flow cytometric method, IFNgamma real time RT-PCR, and IFNgamma ELISPOT for detection of immunologic response to gp100 and MelanA/MART-1 in melanoma patients.
Validation of a HLA-A2 tetramer flow cytometric method, IFNgamma real time RT-PCR, and IFNgamma ELISPOT for detection of immunologic response to gp100 and MelanA/MART-1 in melanoma patients.
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DOI：
10.1186/1479-5876-6-61
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发表时间：
2008-10-22
影响因子：
7.4
通讯作者：
Richards, Susan M.
中科院分区：
文献类型：
--
作者：
Xu, Yuanxin;Theobald, Valerie;Sung, Crystal;DePalma, Kathleen;Atwater, Laura;Seiger, Keirsten;Perricone, Michael A.;Richards, Susan M.
HLA-A2四聚体流式细胞术、IFNγ真实的时间RT-PCR和IFNγ ELISPOT测定通常用作癌症免疫治疗的替代免疫终点。虽然这些通常被用作研究测定来评估患者的免疫反应，但测定验证是必要的，以确保可靠和可重复的结果，并实现更准确的数据解释。在这里，我们描述了一个严格的验证方法，这些检测方法之前，他们用于临床样本分析。建立了各试验的标准操作规程。用从HLA-A2黑色素瘤患者中分离的肿瘤浸润淋巴细胞系（TIL）验证了对gp 100209（210 M）和MART-126-35（27 L）特异的HLA-A2（A*0201）四聚体测定、IFNγ真实的时间RT-PCR和ELISPOT方法。将对gp 100（TIL 1520）或MART-1（TIL 1143和TIL 1235）特异的TIL细胞单独使用或掺入来自健康受试者的冷冻保存的HLA-A2 PBMC中。用肽（gp 100209、gp 100库、MART-127-35或流感病毒-M1和阴性对照肽HIV）刺激TIL/PBMC，以进一步评估真实的时间RT-PCR和ELISPOT方法的测定性能特征。验证参数包括专属性、准确度、精密度、稀释线性、检测限（LOD）和定量限（LOQ）。此外，在正常HLA-A2 PBMC样品中建立了分布。确定了试验对照品的参考范围。验证过程表明，HLA-A2四聚体、IFNγ真实的时间RT-PCR和IFNγ ELISPOT对每种抗原具有高度特异性，gp 100和MelanA/MART-1之间的交叉反应性最小。方法灵敏度高;通过四聚体分析可以在低至1/4545-1/6667的细胞、通过真实的时间RT-PCR可以在1/50，000的细胞和通过ELISPOT可以在1/10，000 -1/20，000的细胞实现检测。尽管已知流式细胞术测定和基于细胞的功能测定具有高测定变异性，但测定符合精确度标准，%CV < 20%（除了使用高PBMC数的ELISPOT，%CV < 25%）。最重要的是，证明了检测试剂盒可有效用于其预期用途。用RT-PCR和ELISPOT方法检测3例黑色素瘤患者PBMC中gp 100的表达。另一名患者显示通过所有3种经验证的方法测量的阳性MART-1应答。我们的结果表明四聚体流式细胞术测定、IFNγ实时RT-PCR和INFγ ELISPOT符合验证标准。验证方法为该领域的其他人提供了指导，以验证这些和其他类似的测定法用于评估患者T细胞应答。这些方法不仅可以应用于癌症疫苗，还可以应用于其他治疗性蛋白质，作为免疫原性和安全性分析的一部分。
HLA-A2 tetramer flow cytometry, IFNγ real time RT-PCR and IFNγ ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patient's immunologic response, assay validation is necessary to ensure reliable and reproducible results and enable more accurate data interpretation. Here we describe a rigorous validation approach for each of these assays prior to their use for clinical sample analysis. Standard operating procedures for each assay were established. HLA-A2 (A*0201) tetramer assay specific for gp100209(210M) and MART-126–35(27L), IFNγ real time RT-PCR and ELISPOT methods were validated using tumor infiltrating lymphocyte cell lines (TIL) isolated from HLA-A2 melanoma patients. TIL cells, specific for gp100 (TIL 1520) or MART-1 (TIL 1143 and TIL1235), were used alone or spiked into cryopreserved HLA-A2 PBMC from healthy subjects. TIL/PBMC were stimulated with peptides (gp100209, gp100pool, MART-127–35, or influenza-M1 and negative control peptide HIV) to further assess assay performance characteristics for real time RT-PCR and ELISPOT methods. Validation parameters included specificity, accuracy, precision, linearity of dilution, limit of detection (LOD) and limit of quantification (LOQ). In addition, distribution was established in normal HLA-A2 PBMC samples. Reference ranges for assay controls were established. The validation process demonstrated that the HLA-A2 tetramer, IFNγ real time RT-PCR, and IFNγ ELISPOT were highly specific for each antigen, with minimal cross-reactivity between gp100 and MelanA/MART-1. The assays were sensitive; detection could be achieved at as few as 1/4545–1/6667 cells by tetramer analysis, 1/50,000 cells by real time RT-PCR, and 1/10,000–1/20,000 by ELISPOT. The assays met criteria for precision with %CV < 20% (except ELISPOT using high PBMC numbers with %CV < 25%) although flow cytometric assays and cell based functional assays are known to have high assay variability. Most importantly, assays were demonstrated to be effective for their intended use. A positive IFNγ response (by RT-PCR and ELISPOT) to gp100 was demonstrated in PBMC from 3 melanoma patients. Another patient showed a positive MART-1 response measured by all 3 validated methods. Our results demonstrated the tetramer flow cytometry assay, IFNγ real-time RT-PCR, and INFγ ELISPOT met validation criteria. Validation approaches provide a guide for others in the field to validate these and other similar assays for assessment of patient T cell response. These methods can be applied not only to cancer vaccines but to other therapeutic proteins as part of immunogenicity and safety analyses.
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影响因子：
3.9
作者：
Speiser, DE;Pittet, MJ;Romero, P
通讯作者：
Romero, P
影响因子：
15.9
作者：
Kaufman, HL;DeRaffele, G;Marincola, FM
通讯作者：
Marincola, FM
影响因子：
3
作者：
Nielsen, MB;Marincola, FM
通讯作者：
Marincola, FM
DOI：
10.1093/jnci/92.16.1336
发表时间：
2000-08-16
期刊：
JOURNAL OF THE NATIONAL CANCER INSTITUTE
影响因子：
--
作者：
Kammula, US;Marincola, FM;Rosenberg, SA
通讯作者：
Rosenberg, SA
影响因子：
4.4
作者：
Dunbar, PR;Smith, CL;Cerundolo, V
通讯作者：
Cerundolo, V