Quantitating MHC Class I Ligand Production and Presentation Using TCR-Like Antibodies.
Quantitating MHC Class I Ligand Production and Presentation Using TCR-Like Antibodies.
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使用 TCR 样抗体定量 MHC I 类配体的产生和呈现。
DOI：
10.1007/978-1-4939-9450-2_12
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Dolan,BrianP
中科院分区：
文献类型：
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作者：
Dolan,BrianP
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- mhc I类复合物的数量是必要的。在这里，我描述了一种定量流式细胞术的应用，用于确定在组织培养中生长的细胞表面的肽- mhc复合物的数量，这些细胞表达肽来源于一种内源性蛋白质。该程序需要一种单克隆抗体，该抗体能够区分呈现感兴趣肽的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珠进行比较，以确定结合在细胞表面的抗体的相对数量，从而确定细胞表达的特定肽- mhc复合物的数量。随着对肽- mhc复合物具有tcr样特异性的新单克隆抗体的产生，该方法将有助于定量细胞类型产生的复合物的确切数量，并将这些数量与T细胞活化的生理结果联系起来。
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.