báo cáo hóa học: 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

Tuyển tập các báo cáo nghiên cứu về hóa học được đăng trên tạp chí sinh học quốc tế đề tài :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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báo cáo hóa học:" 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"Journal of Translational Medicine BioMed Central Open AccessResearchValidation of a HLA-A2 tetramer flow cytometric method,IFNgamma real time RT-PCR, and IFNgamma ELISPOT fordetection of immunologic response to gp100 and MelanA/MART-1in melanoma patientsYuanxin Xu*, Valerie Theobald, Crystal Sung, Kathleen DePalma,Laura Atwater, Keirsten Seiger, Michael A Perricone and Susan M RichardsAddress: Genzyme Corporation, One Mountain Road, Framingham, Massachusetts, MA 01701, USAEmail: Yuanxin Xu* - yuanxin.xu@genzyme.com; Valerie Theobald - valerie.theobald@genzyme.com;Crystal Sung - crystal.sung@genzyme.com; Kathleen DePalma - whaka01@yahoo.com; Laura Atwater - laura.atwater@genzyme.com;Keirsten Seiger - kseiger@comcast.net; Michael A Perricone - michael.perricone@genzyme.com;Susan M Richards - susan.richards@genzyme.com* Corresponding authorPublished: 22 October 2008 Received: 3 October 2008 Accepted: 22 October 2008Journal of Translational Medicine 2008, 6:61 doi:10.1186/1479-5876-6-61This article is available from: http://www.translational-medicine.com/content/6/1/61© 2008 Xu et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: 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 patients 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. Methods: 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. Results: 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 Page 1 of 25 (page number not for citation purposes)Journal of Translational Medicine 2008, 6:61 ...