Tuijnman W B, Van de Winkel J G, Capel P J
Department of Experimental Immunology, University of Utrecht, The Netherlands.
J Immunol Methods. 1990 Mar 9;127(2):207-14. doi: 10.1016/0022-1759(90)90070-c.
We have developed a sensitive and flexible method for the qualitative evaluation of IgG-Fc receptor interactions in cell suspensions. The assay is based on the flow cytometric quantitation of antibody-coated erythrocyte (EA) rosetting using fluorescein-labelled indicator erythrocytes (E). The number of IgG molecules on indicator E, an important parameter in EA rosetting, was estimated by calibrated flow cytometry. EA binding quantitated by this method was correlated with microscopically evaluated rosette formation. Besides automated quantitation of EA binding, this method offers the additional advantage of simultaneously using a second fluorescence parameter, permitting analysis of FcR activity in subpopulations of cells. As an example of the applicability of this approach the binding characteristics of E sensitized with a series of murine heavy chain isotype switch variant monoclonal antibodies against glycophorin A, to the low affinity receptor on K562 cells were determined. Remarkably, the results suggest a comparable affinity of Fc gamma RII on these cells for immunoglobulins of the murine IgG1, IgG2a and IgG2b isotypes.
我们已经开发出一种灵敏且灵活的方法，用于对细胞悬液中IgG-Fc受体相互作用进行定性评估。该测定法基于使用荧光素标记的指示红细胞（E）对抗体包被红细胞（EA）花环形成进行流式细胞术定量。通过校准的流式细胞术估计指示E上的IgG分子数量，这是EA花环形成中的一个重要参数。用这种方法定量的EA结合与显微镜评估的花环形成相关。除了对EA结合进行自动定量外，该方法还具有同时使用第二个荧光参数的额外优势，允许分析细胞亚群中的FcR活性。作为该方法适用性的一个例子，测定了用一系列抗血型糖蛋白A的小鼠重链同种型转换变体单克隆抗体致敏的E与K562细胞上低亲和力受体的结合特性。值得注意的是，结果表明这些细胞上的FcγRII对小鼠IgG1、IgG2a和IgG2b同种型免疫球蛋白具有相当的亲和力。
