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首页> 外文期刊>Biomedical Chromatography: An International Journal Devoted to Research in Chromatographic Methodologies and Their Applications in the Biosciences >Combination of HPLC and solid-phase binding assay for isolation and purification of MHC class I and associated peptides using a bladder tumour cell line.
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Combination of HPLC and solid-phase binding assay for isolation and purification of MHC class I and associated peptides using a bladder tumour cell line.

机译:HPLC和固相结合分析相结合,用于通过膀胱肿瘤细胞系分离和纯化I类MHC和相关肽。

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摘要

The major histocompatibility complex (MHC) class I molecules present processed self and non-self peptides to T lymphocytes. Given that the class I peptide complex plays a critical role in cell-mediated immunity, it is important to identify the nature of class I-associated peptides unique to malignant cells as a prelude to the development of vaccines. The aim of this study was to combine immuno-bead purification (using anti-class I antibody W6/32) technique, sequential ultra-filtration and high performance liquid chromatography (HPLC) to isolate class I antigens and associated peptides from an in-house established bladder tumour cell line (Fen) whose missing class I antigens had been restored by beta2-microglobulin (beta2-m) gene transfaction. The results were as follows: (a) class I antigens could be separated from tumour cell lysate but only from the class I positive Fen cells; (b) treatment of CNBr-W6/32 beads pre-exposed to class I positive Fen lysate and eluted with dissociation agent (mild acid) resulted in the release of more than 20 peptides at an approximate molecular weight of between 700 and 3000 Da based on SDS-PAGE and silver staining analysis; (c) purified and eluted peptides from class I antigens showed distinct peaks when analysed by HPLC. The data presented in this investigation demonstrated the feasibility of isolating class I antigens and associated peptides from a bladder tumour cell line. The extension of these approaches to isolate peptides from tissue tumour biopsies may help the future of vaccine therapy in cancer patients.
机译:主要的组织相容性复合体(MHC)I类分子将加工过的自身和非自身肽呈现给T淋巴细胞。鉴于I类肽复合物在细胞介导的免疫中起关键作用,因此重要的是要确定恶性细胞特有的I类相关肽的性质,作为疫苗开发的前奏。这项研究的目的是结合免疫珠纯化(使用抗I类抗体W6 / 32)技术,顺序超滤和高效液相色谱(HPLC)从内部分离I类抗原和相关肽建立的膀胱肿瘤细胞系(Fen),其丢失的I类抗原已通过beta2-微球蛋白(beta2-m)基因转移得以恢复。结果如下:(a)可以从肿瘤细胞裂解物中分离出I类抗原,但只能从I类阳性Fen细胞中分离; (b)处理CNBr-W6 / 32珠子,该珠子预先暴露于I类阳性Fen裂解物中,并用解离剂(弱酸)洗脱,导致释放了20多种肽,分子量约为700至3000 Da进行SDS-PAGE和银染分析; (c)通过HPLC分析时,来自I类抗原的纯化和洗脱肽显示出明显的峰。该研究中提供的数据证明了从膀胱肿瘤细胞系中分离I类抗原和相关肽的可行性。这些从组织肿瘤活检物中分离肽的方法的扩展可能有助于癌症患者中疫苗治疗的未来。

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