Dolan Brian P
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
Methods Mol Biol. 2013;960:169-177. doi: 10.1007/978-1-62703-218-6_14.
Accurately determining the number of peptide-MHC class I complexes on the cell surface is necessary when evaluating cellular processes or pharmaceuticals that alter the antigen presentation machinery. Here I describe a quantitative flow cytometry application for determining the number of peptide-MHC complexes on the surface of cells grown in tissue culture that express an endogenous protein from which the peptide is derived. The procedure requires a monoclonal antibody with the ability to distinguish MHC class I molecules presenting the peptide of interest from other peptide-MHC complexes. Fluorescence signal measured on antibody-labeled cells can be compared to fluorescent-calibrated beads to determine the relative number of antibodies bound to the cell surface and hence the number of specific peptide-MHC complexes expressed by the cell. As new monoclonal antibodies with TCR-like specificity for peptide-MHC complexes are created, this method will be helpful in quantifying the exact numbers of complexes generated by cell types and relating these numbers to physiological outcomes of T cell activation.
在评估改变抗原呈递机制的细胞过程或药物时，准确测定细胞表面肽 - 主要组织相容性复合体I类（peptide-MHC class I）复合物的数量是必要的。在此，我描述了一种定量流式细胞术应用，用于确定在组织培养中生长的细胞表面上肽 - MHC复合物的数量，这些细胞表达一种内源性蛋白质，而肽正是来源于该蛋白质。该程序需要一种单克隆抗体，该抗体能够区分呈递感兴趣肽的MHC I类分子与其他肽 - MHC复合物。在抗体标记的细胞上测量的荧光信号可以与荧光校准微球进行比较，以确定与细胞表面结合的抗体的相对数量，从而确定细胞表达的特异性肽 - MHC复合物的数量。随着针对肽 - MHC复合物产生具有类似T细胞受体（TCR）特异性的新单克隆抗体，该方法将有助于量化细胞类型产生的复合物的确切数量，并将这些数量与T细胞激活的生理结果相关联。
