Binding of IgG to MoFc gamma RII purified and reconstituted into supported planar membranes as measured by total internal reflection fluorescence microscopy.
Binding of IgG to MoFc gamma RII purified and reconstituted into supported planar membranes as measured by total internal reflection fluorescence microscopy.
复制标题
通过全内反射荧光显微镜测量，IgG 与 MoFc gamma RII 的结合被纯化并重构为支持的平面膜。
DOI：
10.1021/bi00241a005
复制
发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Thompson,NL
中科院分区：
文献类型：
--
作者：
Poglitsch,CL;Sumner,MT;Thompson,NL
化学系，北卡罗来纳州查佩尔山，查佩尔山，北卡罗来纳州27599-3290接收于1990年12月28日;修订版手册接收于1991年4月10日摘要：全内反射荧光显微镜（TIRFM）已与小鼠IgG受体1110 FC 7 RII在底物中的功能重建相结合，支持的平面膜，以定量探测IgG-moFc 7 RII相互作用。从巨噬细胞相关细胞系J774 A纯化M0 FC 7 RII。1使用亲和色谱法，用抗-moFc 7 RII单克隆抗体2.4 G2的Fab片段。通过去污剂透析将纯化的moFc γ RII重构为脂质体，并将脂质体融合在石英基底上以形成含有1110 FC 7 RII的支撑平面膜。TIRFM测量显示荧光标记的2.4 G2 Fab特异性结合平面膜，证实了moFc 7 RII的存在。平面膜中的受体密度足够高以允许用TIRFM直接检测结合的、荧光标记的多克隆和单克隆小鼠IgG，表明在平面膜形成后moFc 7 RII保留Fc介导的IgG结合活性，并允许直接测量结合的IgG作为IgG溶液浓度的函数。交叉抑制实验表明，小鼠IgG多克隆抗体阻断了标记的2.4 G2 Fab的结合，而2.4 G2 Fab则阻断了标记的多克隆抗体的结合。这项工作提供了一个相对较弱的IgG-moFc 7 RII结合常数的直接测量，并证明了一个新的模型系统，其中IgG-moFc 7 RII相互作用的化学和物理性质可以定量表征为膜，抗体和溶液性质的函数。抗体Fc区的0/ell表面受体存在于许多免疫细胞类型，包括巨噬细胞，淋巴细胞，自然杀伤细胞、嗜碱性粒细胞、肥大细胞和多形核白细胞。已经鉴定了一系列具有不同抗体类别和亚类特异性的不同抗体受体。通过连接细胞免疫和体液免疫应答，这些受体在抗体介导的细胞免疫机制如吞噬、B细胞活化和调节、细胞脱粒和抗体依赖性细胞介导的细胞毒性中起关键作用。
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290 Received December 28, 1990; Revised Manuscript Received April 10, 1991 abstract: Total internal reflection fluorescence microscopy (TIRFM) has been combined with functional reconstitution of the mouse IgG receptor 1110FC7RII in substrate-supported planar membranes to quantitatively probe IgG-moFc7RII interactions. M0FC7RII was purified from the macrophage-related cell line J774A. 1 using affinity chromatography with Fab fragments of the anti-moFc7RII monoclonal antibody 2.4 G2. Purified moFcyRII was reconstituted into liposomes by detergent dialysis, and the liposomes were fused on quartz substrates to form supported planar membranes containing 1110FC7RII. TIRFM measurements showed that fluorescently labeled 2.4 G2 Fab specifically bound to the planar membranes, confirming the presence of moFc7RII. The receptor density in the planar membranes was sufficiently highto allow direct detection of bound, fluorescently labeled polyclonal and monoclonal mouse IgG with TIRFM, demonstrating that moFc7RII retained Fc-mediated IgG binding activity after planarmembrane formation and permitting direct measurement of bound IgG as a function of the IgG solution concentration. Cross-inhibition mea-surements showed that polyclonal mouse IgG blockedthe binding of labeled 2.4 G2 Fab and that 2.4 G2 Fab blocked the binding of labeled polyclonal IgG. Thiswork provides a direct measure of the relatively weak IgG-moFc7RII association constant and demonstrates a new model system in which the chemical and physical properties of IgG-moFc7RII interactions can be quantitatively characterized as a function of membrane, antibody, and solution properties.0/ell surface receptors for the Fc region of antibodies are found on a number of immunologicalcell types, including macrophages, lymphocytes, natural killer cells, basophils, mast cells, and polymorphonuclear leukocytes. A range of different antibody receptors with distinct antibody class and subclass specificities have been identified. By linking the cellular and humoral immune responses, these receptors play key roles in antibody-mediated cellular immune mechanismssuch as phagocytosis, B cell activation and regulation, cellular de-granulation, and antibody-dependent cell-mediated cytotox-icity.