Gulle H, Eibl M M, Wolf H M
Institute of Immunology, University of Vienna, Austria.
J Immunol Methods. 1998 May 1;214(1-2):199-208. doi: 10.1016/s0022-1759(98)00058-1.
Nitrocellulose (NC) has proved to be a versatile tool for the isolation and characterization of various biomolecules. In this report we extend its scope by using antibody-coated NC particles to cross-link molecules on the surface of living cells. Ligation of receptors in Jurkat cells with NC-bound specific antibodies induced protein tyrosine phosphorylation patterns of cellular proteins comparable to conventional antibody cross-linking. In addition, the present study shows that application of NC particles coated with human IgA significantly activated monocytic cells via the Fc alpha receptor (Fc alphaR), whereas cross-linking of receptor-ligand complexes with isotype-specific antibody was less efficient. Subsequent immunoprecipitation and immunoblot analysis of aggregated Fc receptors (FcRs) complexed to Ig-adsorbed particles permits fast identification of molecules involved in the transmission of signals. Therefore, ligand-coated NC particles can be used to examine receptor-mediated cell activation events dependent upon extensive receptor aggregation.
硝化纤维素（NC）已被证明是用于分离和表征各种生物分子的多功能工具。在本报告中，我们通过使用抗体包被的NC颗粒交联活细胞表面的分子来扩展其应用范围。用与NC结合的特异性抗体连接Jurkat细胞中的受体，可诱导细胞蛋白的蛋白质酪氨酸磷酸化模式，这与传统抗体交联相当。此外，本研究表明，用人IgA包被的NC颗粒通过Fcα受体（FcαR）显著激活单核细胞，而异型特异性抗体交联受体-配体复合物的效率较低。随后对与Ig吸附颗粒复合的聚集Fc受体（FcR）进行免疫沉淀和免疫印迹分析，可快速鉴定参与信号转导的分子。因此，配体包被的NC颗粒可用于检测依赖于广泛受体聚集的受体介导的细胞激活事件。
