Zeh H J, Leder G H, Lotze M T, Salter R D, Tector M, Stuber G, Modrow S, Storkus W J
Department of Surgery, University of Pittsburgh, Pennsylvania.
Hum Immunol. 1994 Feb;39(2):79-86. doi: 10.1016/0198-8859(94)90105-8.
A novel class I-peptide-binding assay was developed and used to identify a series of peptides derived from the human p53 tumor-suppressor gene product capable of binding the HLA-A2 class I allele. Brief pH 3.3 acid treatment of human cell lines rapidly denatures pre-existing class I complexes, as detected by loss of binding of conformation-dependent mAbs, leaving only free class I heavy chains associated with the viable cell surface. These heavy chains may be induced to refold and be recognized by antibodies (in 2-4 hours) when acid-treated cells are coincubated with exogenous beta 2-microglobulin and peptides capable of binding the relevant class I allele examined. This assay, with a detection limit of 1-10 nM peptide, was used to screen the capacity of a panel of nine peptides bearing HLA-A2-binding motifs and derived from the human p53 tumor-suppressor protein sequence. Eight of the nine peptides bound to, and reconstituted, HLA-A2 on acid-treated cells. This assay system will enable the rapid identification of peptides binding to any class I allele, which is the initial prerequisite for elucidating potential CD8+ T-cell epitopes.
开发了一种新型的I类肽结合测定法，并用于鉴定一系列源自人p53肿瘤抑制基因产物且能够结合HLA - A2 I类等位基因的肽。如通过构象依赖性单克隆抗体结合丧失所检测到的，对人细胞系进行短暂的pH 3.3酸处理会使预先存在的I类复合物迅速变性，仅留下与活细胞表面相关的游离I类重链。当酸处理的细胞与外源性β2 -微球蛋白以及能够结合所检测的相关I类等位基因的肽共同孵育时，这些重链可能会被诱导重新折叠并在2 - 4小时内被抗体识别。该测定法的检测限为1 - 10 nM肽，用于筛选一组九个带有HLA - A2结合基序且源自人p53肿瘤抑制蛋白序列的肽的结合能力。九个肽中的八个与酸处理细胞上的HLA - A2结合并重构。该测定系统将能够快速鉴定与任何I类等位基因结合的肽，这是阐明潜在CD8 + T细胞表位的首要前提条件。
