Petty H R, Dereski W
Biochemistry. 1985 Jul 16;24(15):4141-8. doi: 10.1021/bi00336a049.
A fluorescein- and lactoperoxidase-conjugated ferritin-anti-ferritin immune complex has been prepared for cell surface labeling experiments on immune recognition and effector function. Lactoperoxidase (LPO) has been covalently coupled to affinity-purified anti-ferritin antibodies with p-benzoquinone by a modified version of the method of Ternynck and Avrameas [Ternynck, T., & Avrameas, S. (1976) Ann. Immunol. (Paris) 127C, 197]. The conjugate is a heterodimer of Mr230 000 with linkages to either or both of the heavy and light chains of the antibody, as judged by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence and presence of 2-mercaptoethanol. The conjugate retains antibody-binding activity as measured by a quantitative precipitin assay. When incorporated into immune complexes, the modified antibody also retains Fc receptor recognition ability as determined by erythrocyte-antibody rosette inhibition assays. Electron microscopy demonstrated that the antigen, ferritin, was monodisperse with complete apoprotein sheaths surrounding the core. Ferritin-anti-ferritin-LPO complexes were formed in 4-fold antigen excess. Complexes were verified by fluorescence and electron microscopy. Immune complexes were masked with "cold" iodine by use of the endogenous LPO activity. The complexes bound to cells at 4 degrees C as shown by electron microscopy and fluorescence video/intensification microscopy. The LPO delivered to the cell surface in this fashion can be utilized to iodinate the surface with 125I. Under saturation conditions, the labeling with local LPO delivery followed by SDS-PAGE and autoradiography is identical with labeling with free LPO. Labeling has also been conducted under conditions of substrate deficit.(ABSTRACT TRUNCATED AT 250 WORDS)
已制备了一种荧光素和乳过氧化物酶偶联的铁蛋白 - 抗铁蛋白免疫复合物，用于免疫识别和效应功能的细胞表面标记实验。通过Ternynck和Avrameas方法的改进版本[Ternynck, T., & Avrameas, S. (1976) Ann. Immunol. (Paris) 127C, 197]，用对苯醌将乳过氧化物酶（LPO）共价偶联到亲和纯化的抗铁蛋白抗体上。通过在有无2 - 巯基乙醇的情况下进行二维十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）判断，该偶联物是Mr230 000的异二聚体，与抗体的重链和轻链之一或两者相连。通过定量沉淀试验测定，该偶联物保留抗体结合活性。当掺入免疫复合物中时，通过红细胞 - 抗体玫瑰花结抑制试验确定，修饰后的抗体也保留Fc受体识别能力。电子显微镜显示，抗原铁蛋白是单分散的，核心周围有完整的脱铁蛋白鞘。在抗原过量4倍的情况下形成铁蛋白 - 抗铁蛋白 - LPO复合物。通过荧光和电子显微镜验证复合物。利用内源性LPO活性用“冷”碘掩盖免疫复合物。如电子显微镜和荧光视频/增强显微镜所示，复合物在4℃下与细胞结合形成。以这种方式递送到细胞表面的LPO可用于用125I对表面进行碘化。在饱和条件下，用局部LPO递送后进行SDS - PAGE和放射自显影的标记与用游离LPO标记相同。标记也在底物不足的条件下进行。（摘要截短于250字）
