Vanni Irene, Ugolotti Elisabetta, Larghero Patrizia, Biassoni Roberto
Molecular Medicine, Translational Medicine Department, Istituto Giannina Gaslini, Via G. Gaslini 5, Genoa, 16147, Italy.
Methods Mol Biol. 2015;1315:133-51. doi: 10.1007/978-1-4939-2715-9_11.
Usually, HLA typing has been performed either by serology-based typing incubating a panel of known anti-HLA antibodies with viable lymphocytes of unknown HLA type or by molecular typing including medium-resolution HLA typing by Sequence Specific Oligonucleotide Probes (SSOP) or high-resolution HLA typing by Sequence Based Typing (SBT). Traditionally, HLA antigens have been defined using serological techniques, but these methods have several disadvantages, such as low resolution, the requirement for viable cells, and cell surface expression of HLA molecules. HLA type screening methods are categorized as low, medium, and high resolution, and only sequencing-based typing methods provide the highest resolution and are considered the gold standard for HLA typing.Among the HLA SBT based-methods, the Pyrosequencing(®) technique is an extremely versatile and accurate real-time sequencing technique with some advantages compared to classic Sanger method.Here, we describe a quick and inexpensive method that allows through the use of Pyrosequencing subtyping of HLA class I molecules, into HLA-Bw6, -Bw4 I80, or -Bw4 T80 and HLA-C1, or -C2 groups. In particular, this analysis is focused on the amino acids around residue 80. This method demonstrated good sensitivity, specificity, and reproducibility. Using a quantitative allele acquisition mode, the method provides accurate sequence information required for the definition of heterozygous and/or homozygous samples.
通常，HLA分型可通过基于血清学的分型方法进行，即将一组已知的抗HLA抗体与未知HLA类型的活淋巴细胞进行孵育，也可通过分子分型方法进行，包括使用序列特异性寡核苷酸探针（SSOP）进行中等分辨率的HLA分型，或使用基于序列的分型（SBT）进行高分辨率的HLA分型。传统上，HLA抗原是使用血清学技术来定义的，但这些方法存在一些缺点，如分辨率低、需要活细胞以及HLA分子的细胞表面表达。HLA分型筛选方法分为低、中、高分辨率，只有基于测序的分型方法提供最高分辨率，被认为是HLA分型的金标准。在基于HLA SBT的方法中，焦磷酸测序技术是一种极其通用且准确的实时测序技术，与经典的桑格法相比具有一些优势。在此，我们描述了一种快速且廉价的方法，该方法通过使用焦磷酸测序对HLA I类分子进行亚型分析，可将其分为HLA - Bw6、- Bw4 I80或- Bw4 T80以及HLA - C1或- C2组。特别地，该分析聚焦于第80位残基周围的氨基酸。该方法显示出良好的敏感性、特异性和可重复性。使用定量等位基因获取模式，该方法可提供定义杂合和/或纯合样本所需的准确序列信息。
