Wilson M R, Mulligan S P, Raison R L
Clinical Immunology Research Centre, University of Sydney, NSW, Australia.
J Immunol Methods. 1988 Mar 16;107(2):231-7. doi: 10.1016/0022-1759(88)90223-2.
The screening of panels of hybridoma supernatants for specific secreted monoclonal antibodies is often achieved by cellular immunofluorescent staining and flow cytometric analysis. In some circumstances such assays are difficult because the required antigen-bearing cell population is not suitable for use in flow cytometry, has limited cell cycle expression or poor in vitro growth. A method is presented here that provides a solution to these difficulties. A system was developed, using polyacrylamide microspheres coupled with cell membranes, which permitted the production of an easily stored, standardised antigen source which could be used in subsequent flow cytometric assays. Studies comparing the binding of antibodies to whole cells and cell membrane-coupled microspheres indicate a strong qualitative and quantitative correlation in the expression of surface antigens. It is shown here that membrane antigens can be stored coupled to microspheres for months and still retain good reactivity with the appropriate antibodies. This same technique could be used in studies of antigens other than those of the mammalian cell membrane - for example, membrane antigens of sub-cellular organelles such as the mitochondrion and endoplasmic reticulum, plant or bacterial membrane antigens, or antigens not associated with membranes such as viral proteins.
通过细胞免疫荧光染色和流式细胞术分析，常常可以实现对杂交瘤细胞上清液中特异性分泌的单克隆抗体进行筛选。在某些情况下，此类检测会遇到困难，因为所需的携带抗原的细胞群体不适合用于流式细胞术分析，其细胞周期表达有限或体外生长能力较差。本文介绍了一种解决这些难题的方法。我们开发了一种系统，利用与细胞膜偶联的聚丙烯酰胺微球，从而能够制备出易于储存的标准化抗原源，可用于后续的流式细胞术检测。比较抗体与完整细胞以及与细胞膜偶联微球结合情况的研究表明，表面抗原的表达在定性和定量方面都具有很强的相关性。本文证明，膜抗原与微球偶联后可储存数月，并且仍然能够与相应抗体保持良好的反应性。这项技术同样可用于研究除哺乳动物细胞膜抗原之外的其他抗原，例如线粒体和内质网等亚细胞器的膜抗原、植物或细菌膜抗原，或者与膜无关的抗原，如病毒蛋白。
