Peptide Binding to HLA Class I Molecules: Homogenous, High-Throughput Screening, and Affinity Assays
Peptide Binding to HLA Class I Molecules: Homogenous, High-Throughput Screening, and Affinity Assays
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DOI：
10.1177/1087057108329453
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发表时间：
2009-02-01
影响因子：
--
通讯作者：
Buus, Soren
中科院分区：
文献类型：
--
作者：
Harndahl, Mikkel;Justesen, Sune;Buus, Soren
人类MHC项目的目标是大规模描述多肽--与广泛的人类白细胞抗原分子结合，覆盖全球所有人群，以及随之而来的能够预测任何给定的多肽与任何给定的人类白细胞抗原分子结合的生物信息学工具。在这里，作者提出了一种均相的、基于邻近性的分析方法，用于检测与HLAI类分子结合的多肽。它使用构象依赖的抗HLAI类抗体W6/32作为一个标签，生物素化的重组HLAI类分子作为另一个标签，通过发光氧通道免疫分析技术(简称LOCAL，商业化名称为AlphaScreen(TM))产生基于邻近的信号。与基于酶联免疫吸附分析的多肽-人类白细胞抗原I类结合分析相比，该基因座分析产生了几乎相同的亲和力测量，尽管具有更宽的动态范围、更好的信号与背景比和更高的容量。他们还描述了一种筛选与人类白细胞抗原分子结合的多肽的有效方法。对于偶尔使用的人来说，这将是一种可靠、简单的多肽-人类白细胞抗原结合试验。对于更专注的用户，它可以使用标准液体处理机器人和384孔板在高通量筛选模式下轻松执行。我们已经成功地将这种分析应用于60多种不同的人类白细胞抗原分子，导致了200多万次测量。(生物分子筛选杂志2009：173-180)
The Human MHC Project aims at large-scale description of peptide-HLA binding to a wide range of HLA molecules covering all populations of the world and the accompanying generation of bioinformatics tools capable of predicting binding of any given peptide to any given HLA molecule. Here, the authors present a homogenous, proximity-based assay for detection of peptide binding to HLA class I molecules. It uses a conformation-dependent anti-HLA class I antibody, W6/32, as one tag and a biotinylated recombinant HLA class I molecule as the other tag, and a proximity-based signal is generated through the luminescent oxygen channeling immunoassay technology (abbreviated LOCI and commercialized as AlphaScreen (TM)). Compared with an enzyme-linked immunosorbent assay-based peptide-HLA class I binding assay, the LOCI assay yields virtually identical affinity measurements, although having a broader dynamic range, better signal-to-background ratios, and a higher capacity. They also describe an efficient approach to screen peptides for binding to HLA molecules. For the occasional user, this will serve as a robust, simple peptide-HLA binding assay. For the more dedicated user, it can easily be performed in a high-throughput screening mode using standard liquid handling robotics and 384-well plates. We have successfully applied this assay to more than 60 different HLA molecules, leading to more than 2 million measurements. (Journal of Biomolecular Screening 2009: 173-180)