Zola H, Neoh S H, Mantzioris B X, Webster J, Loughnan M S
Department of Clinical Immunology, Flinders Medical Centre Bedford Park, Australia.
J Immunol Methods. 1990 Dec 31;135(1-2):247-55. doi: 10.1016/0022-1759(90)90278-4.
Receptors for lymphokines and growth factors are present on cell surfaces often at concentrations of 100-500 copies per cell. Although conventional immunofluorescence cannot detect such low levels, cell membrane antigens present at these concentrations can be detected using an optimally set up flow cytometer together with a three-layer immunofluorescence technique, consisting of monoclonal antibody reacted with selected batches of biotinylated horse anti-mouse immunoglobulin and phycoerythrin-streptavidin. In this study we purified and radiolabelled a number of monoclonal antibodies, determined the specific radioactivity by self-displacement analysis, and used the radiolabelled antibody in experiments where the number of molecules of antibody bound per cell and the fluorescence intensity were measured on the same sample. This permitted us to determine the sensitivity of the fluorescence procedure in molecules per cell, using several different antibody/target cell combinations. The method was consistently capable of detecting fewer than 100 molecules of antibody bound per cell.
淋巴因子和生长因子的受体通常以每个细胞100 - 500个拷贝的浓度存在于细胞表面。虽然传统的免疫荧光法无法检测到如此低的水平，但使用优化设置的流式细胞仪以及三层免疫荧光技术，可以检测出以这些浓度存在的细胞膜抗原，该技术由单克隆抗体与选定批次的生物素化马抗小鼠免疫球蛋白和藻红蛋白 - 链霉亲和素反应组成。在本研究中，我们纯化并放射性标记了多种单克隆抗体，通过自置换分析确定了比放射性，并在实验中使用放射性标记的抗体，在同一样本上测量每个细胞结合的抗体分子数和荧光强度。这使我们能够使用几种不同的抗体/靶细胞组合，以每个细胞中的分子数来确定荧光程序的灵敏度。该方法始终能够检测到每个细胞结合的抗体分子数少于100个。
