Quantifying aggregation of IgE-FcεRI by multivalent antigen
Quantifying aggregation of IgE-FcεRI by multivalent antigen
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DOI：
10.1016/s0006-3495(99)77397-2
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发表时间：
1999-05-01
影响因子：
3.4
通讯作者：
Posner, RG
中科院分区：
文献类型：
--
作者：
Hlavacek, WS;Perelson, AS;Posner, RG
多价配体对细胞表面受体的聚集可引发多种细胞反应。一个研究得很好的对聚集作出反应的受体是IgE的高亲和力受体(Fc Epsilon RI)，它负责启动过敏反应。为了定量抗原诱导的IgE-Fc epsilon RI复合体的聚集，我们发展了一种基于多参数流式细胞术的方法来监测表面IgE结合位点的占有率和抗原与细胞表面的结合。结合的IgE结合位点的数量超过结合抗原的数量，即受体之间的桥接数量，提供了一种定量测量IgE-Fc epsilon RI聚集的方法。我们用我们的方法研究了半抗原荧光蛋白2，4-二硝基苯酚偶联B-藻红蛋白(DNP25-PE)与异硫氰酸荧光素标记的抗DNP IgE在大鼠嗜碱性白血病细胞表面的平衡结合。结果表明，Ig E-FceRI聚集是如何依赖于DNP25-PE和表面Ig E的总浓度的。正如预期的那样，我们发现最大的聚集发生在一个最佳的抗原浓度。我们还发现聚集性随表面IgE的总浓度而变化，正如先前的理论分析所预测的那样。
Aggregation of cell surface receptors by multivalent ligand can trigger a variety of cellular responses. A well-studied receptor that responds to aggregation is the high affinity receptor for IgE (Fc epsilon RI), which is responsible for initiating allergic reactions. To quantify antigen-induced aggregation of IgE-Fc epsilon RI complexes, we have developed a method based on multiparameter flow cytometry to monitor both occupancy of surface IgE combining sites and association of antigen with the cell surface. The number of bound IgE combining sites in excess of the number of bound antigens, the number of bridges between receptors, provides a quantitative measure of IgE-Fc epsilon RI aggregation. We demonstrate our method by using it to study the equilibrium binding of a haptenated fluorescent protein, 2,4-dinitrophenol-coupled B-phycoerythrin (DNP25-PE), to fluorescein isothiocyanate-labeled anti-DNP IgE on the surface of rat basophilic leukemia cells. The results, which we analyze with the aid of a mathematical model, indicate how IgE-FceRI aggregation depends on the total concentrations of DNP25-PE and surface IgE. As expected, we find that maximal aggregation occurs at an optimal antigen concentration. We also find that aggregation varies qualitatively with the total concentration of surface IgE as predicted by an earlier theoretical analysis.