Paden C M, Hapner S J
Department of Biology, Montana State University, Bozeman 59717.
Brain Res. 1991 Apr 5;545(1-2):151-63. doi: 10.1016/0006-8993(91)91281-5.
Immunocytochemical and immunoblotting technique have been used to characterize the antigens recognized by two monoclonal antibodies (MAbs C6 and D5) produced against dissociated cells from punches of neonatal supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei of the rat. Peroxidase immunocytochemistry revealed that both MAbs label magnocellular perikarya in the adult and neonatal SON and PVN as well as smaller neurons in the suprachiasmatic nucleus. Axons of the hypothalamo-neurohypophysial tract are also immunolabeled within the hypothalamus and zona interna of the median eminence, and C6 and D5 each bind specifically to both the adult and neonatal neurohypophysis. Dual-label immunofluorescence experiments employing C6 or D5 simultaneously with rabbit antisera specific for either oxytocin, neurophysin or vasopressin neurophysin revealed that C6 binds only to vasopressinergic magnocellular perikarya in the SON, while D5 labels both vasopressinergic and a small subset of oxytocinergic magnocellular neurons. Post-embedding immunogold analysis of MAb binding to the neurohypophysis at the ultrastructural level showed that both C6 and D5 recognize antigens associated with large dense core neurosecretory granules in a subset of neurosecretory axons. Initial biochemical characterization of the antigens recognized by C6 and D5 was performed using SDS-PAGE and Western immunoblotting. MAbs C6 and D5 label single protein bands with apparent molecular weights of 38 and 68 kDa, resp., in blots of reduced extracts from the adult neurointermediate lobe. No cross-reactivity between C6 and D5 and the neurophysins was apparent, nor did anti-neurophysin sera recognize the bands identified by C6 and D5. We have therefore designated these novel antigens as VPGP38 and VPGP68 for VasoPressin Granule Proteins.
免疫细胞化学和免疫印迹技术已被用于鉴定由两种单克隆抗体（MAb C6和D5）识别的抗原，这两种抗体是针对从新生大鼠视上核（SON）和室旁核（PVN）下丘脑核团的打孔解离细胞产生的。过氧化物酶免疫细胞化学显示，这两种单克隆抗体在成年和新生的SON和PVN中标记大细胞神经元胞体，以及视交叉上核中的较小神经元。下丘脑-神经垂体束的轴突在下丘脑和正中隆起的内侧区内也被免疫标记，并且C6和D5各自特异性结合成年和新生神经垂体。使用C6或D5与针对催产素、神经垂体素或血管加压素神经垂体素的兔抗血清同时进行的双标记免疫荧光实验显示，C6仅与SON中表达血管加压素的大细胞神经元胞体结合，而D5标记表达血管加压素的大细胞神经元以及一小部分表达催产素的大细胞神经元。在超微结构水平上对单克隆抗体与神经垂体结合进行的包埋后免疫金分析表明，C6和D5都识别神经分泌轴突亚群中与大的致密核心神经分泌颗粒相关的抗原。使用SDS-PAGE和Western免疫印迹对C6和D5识别的抗原进行了初步生化鉴定。在成年神经中间叶还原提取物的印迹中，单克隆抗体C6和D5分别标记表观分子量为38 kDa和68 kDa的单一蛋白条带。C6和D5与神经垂体素之间没有明显的交叉反应，抗神经垂体素血清也未识别由C6和D5鉴定的条带。因此，我们将这些新抗原命名为血管加压素颗粒蛋白VPGP38和VPGP68。
