Monoclonal antibody to soluble guanylate cyclase of rat brain
Monoclonal antibody to soluble guanylate cyclase of rat brain
复制标题
大鼠脑可溶性鸟苷酸环化酶单克隆抗体
DOI：
10.1016/0014-5793(82)80527-9
复制
发表时间：
1982
期刊：
影响因子：
3.5
通讯作者：
T. Deguchi
中科院分区：
文献类型：
--
作者：
M. Nakane;T. Deguchi
鸟苷酸环化酶（GTP焦磷酸裂解酶，环化，EC 4.6。1.2），负责环GMP合成的酶，主要在哺乳动物组织的可溶性部分中检测到，部分与颗粒部分相关[1 -61]。在大鼠脑中，鸟苷酸环化酶主要存在于突触体可溶性部分中，表明其参与神经传递[6]。我们已经从牛脑中纯化了可溶性鸟苷酸环化酶，使其具有明显的同质性，并通过免疫家兔制备了该酶的抗体[7]。该抗体有效地抑制可溶性鸟苷酸环化酶活性从各种哺乳动物组织，但不是从组织的Triton分散颗粒鸟苷酸环化酶。已经建立了制备单克隆抗体的方法[8]。由于其高度特异性，单克隆抗体将提供用于研究酶分子的不同性质和用于组织化学证明鸟苷酸环化酶的细胞以及亚细胞定位的方法。本文报道了抗大鼠脑可溶性鸟苷酸环化酶单克隆抗体的制备及其性质。在无百日咳微生物的情况下，以3周间隔进行两次加强注射。在最后一次加强注射后3天从免疫的小鼠中取出脾细胞，并如[9]中所述与小鼠骨髓瘤细胞P3-NS I/l-Ag 4-l（NS I）融合。通过加入1 ml 50%聚乙二醇4000，将脾细胞（1.6 × 108）与4 × 107骨髓瘤细胞融合。将细胞悬浮在生长培养基（补充有15%胎牛血清、2 mM L-谷氨酰胺和ImM丙酮酸盐的RPM 11640）中，并接种在两个96孔Nunc Microtest板中。两天后，加入0.1ml HAT选择性培养基（生长培养基补充有100 μ M次黄嘌呤、0.4 μ M氨基蝶呤和16 μ M胸苷），在随后的10天内将一半培养基更换3次。细胞融合后14天，通过如下所述测量酶抑制来测定培养液（60 μ l）的抗鸟苷酸环化酶抗体。通过重复两次的有限稀释法获得单克隆杂交瘤细胞系。
Guanylate cyclase (GTP pyrophosphate-lyase, cyclizing, EC 4.6. 1.2), the enzyme responsible for the synthesis of cyclic GMP, is detected mostly in soluble fraction and partly associated with particulate fraction of mammalian tissues [l-61. In rat brain, guanylate cyclase is found mainly in synaptosomal soluble fraction suggesting its involvement in the neural transmission [6]. We have purified the soluble guanyiate cyclase from bovine brain to an apparent homogeneity and produced the antibody to the enzyme by immunizing a rabbit [7]. The antibody effectively inhibited the soluble guanylate cyclase activity from various mammalian tissues, but not the Triton-dispersed particulate guanylate cyclase from tissues. A method for preparing a monoclonal antibody has been established [8]. Because of its high specificity, a monoclonal antibody would offer a method for studying the different properties of enzyme molecules and for histochemically demonstrating the cellular as well as subcellular localization of guanylate cyclase. We report here production and properties of the monoclonal antibody to the soluble guanylate cyclase of rat brain.The purified guanylate cyclase (64OMg) was injected into BALB/c mouse intraperitoneally with 2 X lo9 heat-inactivated pertussis organisms. The booster injections were carried out twice at a 3-week interval without pertussis organisms. Spleen cells were taken from immunized mouse 3 days after the last booster injection and fused with mouse myeloma cells P3-NS I/l-Ag4-1 (NS I) as in [9]. Spleen cells (1.6 X 108) were fused with 4 X 10’myeloma cells by the addition of 1 ml 50% polyethylene glycol4000. The cells were suspended in growth medium (RPM1 1640 supplemented with 15% fetal bovine serum, 2 mM L-glutamine and 1 mM pyruvate) and inoculated in two 96-well Nunc Microtest Plates. Two days later, 0.1 ml of HAT selective medium (growth medium supplemented with 100 PM hypoxanthine, 0.4 PM aminopterin and 16 PM thymidine) was added and half of the medium was replaced 3 times during the subsequent 10 days. Fourteen days after cell fusion, culture fluid (60 fl) was assayed for anti-guanylate cyclase antibody by measuring enzyme inhibition as described below. Monoclonal hybridoma cell lines were obtainec’by a limiting dilution method repeated twice.