Chang L, Kjer-Nielsen L, Flynn S, Brooks A G, Mannering S I, Honeyman M C, Harrison L C, McCluskey J, Purcell A W
Department of Microbiology and Immunology and ImmunoID, University of Melbourne, Vic., Australia.
Tissue Antigens. 2003 Nov;62(5):408-17. doi: 10.1034/j.1399-0039.2003.00122.x.
We describe a strategy for identifying ligands of human leukocyte antigen (HLA) class I molecules based on a peptide library-mediated in vitro assembly of recombinant class I molecules. We established a microscale class I assembly assay and used a capture ELISA to quantify the assembled HLA-peptide complexes. The identity of the bound ligands was then deduced by mass spectrometry. In this method, HLA complexes assembled in vitro in the presence of components of a mixture of peptides were immunoprecipitated and the bound peptide(s) identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. This process of epitope extraction is robust and can be used with complex mixtures containing in excess of 300 candidate ligands. A library of overlapping peptides representing all potential octamers, nonamers and decamers from human preproinsulin was synthesized using unique library chemistry. Peptides from the library were used to initiate assembly of recombinant HLA-B8, HLA-B15 and HLA-A2, facilitating the identification of candidate T-cell epitopes from preproinsulin.
我们描述了一种基于肽库介导的重组I类分子体外组装来鉴定人类白细胞抗原（HLA）I类分子配体的策略。我们建立了一种微量I类组装测定法，并使用捕获酶联免疫吸附测定（ELISA）来定量组装的HLA-肽复合物。然后通过质谱推断结合配体的身份。在该方法中，在肽混合物的成分存在下体外组装的HLA复合物被免疫沉淀，并通过基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱鉴定结合的肽。这种表位提取过程是可靠的，可用于含有超过300种候选配体的复杂混合物。使用独特的文库化学合成了一个代表来自人胰岛素原前体的所有潜在八聚体、九聚体和十聚体的重叠肽文库。来自该文库的肽用于启动重组HLA-B8、HLA-B15和HLA-A2的组装，有助于从胰岛素原前体中鉴定候选T细胞表位。
