Chersi A, Garzillo C, Butler R H, Tanigaki N
Regina Elena Institute for Cancer Research, Rome, Italy.
Hum Immunol. 2001 Aug;62(8):858-68. doi: 10.1016/s0198-8859(01)00273-7.
Allelic variations of in vitro HLA class I assembly have been investigated in both the absence and the presence of binding peptides by flow cytometry using human leukocyte antigen (HLA) class I alpha chains isolated by alkali treatment from cultured HLA homozygous B cells and polystyrene beads coated with anti-HLA class I alpha chain antibodies specific to the C-terminal segment (anti-HLA class I beads). The specificity of assembly was temperature dependent, while the stability of the assembled complex depended on the bound peptide. The efficiency of assembly was allele dependent and primarily ruled by the binding affinity of alpha chains with beta(2)m. Thus, an allele hierarchy could be defined for the binding of HLA-B alpha chain with beta(2)-microglobulin: B7, B18 > B35, B62 > B27, B51. Allele and temperature dependency was found in HLA class I reassembly on acid treated B cells. The HLA class I proteins, reassembled with specific single peptides, could be efficiently transferred to anti-HLA class I beads. These findings would be used to produce microspheres coupled at high surface density with oriented single-peptide loaded HLA class I molecules and also to improve the preparation efficiency of HLA class I tetramers by the use of site-specific biotinylation.
通过流式细胞术，在不存在和存在结合肽的情况下，研究了体外HLA I类组装的等位基因变异。使用通过碱处理从培养的HLA纯合B细胞中分离的人白细胞抗原（HLA）I类α链和包被有针对C末端片段的抗HLA I类α链抗体的聚苯乙烯珠（抗HLA I类珠）。组装的特异性取决于温度，而组装复合物的稳定性取决于结合的肽。组装效率取决于等位基因，主要由α链与β2微球蛋白的结合亲和力决定。因此，可以定义HLA - B α链与β2 - 微球蛋白结合的等位基因层次结构：B7、B18 > B35、B62 > B27、B51。在酸处理的B细胞上的HLA I类重新组装中发现了等位基因和温度依赖性。用特定的单肽重新组装的HLA I类蛋白可以有效地转移到抗HLA I类珠上。这些发现将用于制备以高表面密度偶联有定向单肽负载的HLA I类分子的微球，也用于通过使用位点特异性生物素化提高HLA I类四聚体的制备效率。
