Neuropathological Diagnosis of Pulp Disease Using Antimyelin-protein Monoclonal Antibody

P0蛋白是一种完整的膜糖蛋白，其占哺乳动物周围神经中总髓鞘蛋白的50-60%。在牙髓病的神经病理学检查中，该糖蛋白可作为标记物。本研究旨在从小分子材料中分离纯化P0糖蛋白。采用蔗糖密度梯度离心法，从大鼠腋神经、肌皮神经、正中神经、桡神经、尺神经和坐骨神经中提取髓鞘组分。用电子显微镜检查这些髓鞘组分。髓鞘组分主要由板层结构组成。亚细胞的细胞器，如细胞核和线粒体，没有观察到在这一部分。将髓磷脂级分在0.03M HCl中均质化，在4 ° C下搅拌并离心。将酸不溶性残留物直接用氯仿/再乙醇匀浆。将无脂肪沉淀物溶解于含有10%十二烷基硫酸钠（SDS）的0.1 M磷酸盐缓冲液中。将上清液上样到Sephacryl S-100柱上，用含有2%SDS的0.1M磷酸盐缓冲液平衡和洗脱。检测到一个主峰。浓缩来自主峰的级分。SDS-聚丙烯酰胺凝胶电泳后，蛋白质用考马斯亮蓝染色，糖蛋白用过碘酸-希夫试剂染色，主峰为单一条带（约32 KD）。结果表明，本研究分离纯化的蛋白质为P0糖蛋白，浓缩后的样品可直接作为制备抗P0糖蛋白单克隆抗体的抗原。

The P0 protein is an integral membrane glycoprotein which comprises 50-60% of the total myelin proteins in mammalian peripheral nerves. Then, in the neuropathological examination of pulp diseases, this glycoprotein could be used as the marker. This study was designed to isolate and purify P0 glycoprotein from a small material. The myelin fractions were prepared from axillar, musculocutaneous, median, radial, ulnar and sciatic nerves of rats by sucrose density gradient centrifugation. This myelin fractions were examined with electron microscope. The myelin fraction consisted mainly of the lammellar structure. Subcellular organelles, such as nuclei and mito chondria, were not observed in this fraction. The myelin fraction was homogenized in 0.03 M HC1, stirred at 4ﾟC and centrifuged. The acid-insoluble residue was homogenized directly with chloroform/reethanol. The fat-free precipitate was dissolved in 0.1 M phosphate buffer containing 10% sodium dodecyl sulfate (SDS). The supernatant was applied on a Sephacryl S-100 column, equilibrated and eluted with 0.1 M phosphate buffer containing 2% SDS. One major peak was detected. The fractions from major peak was concentrated. This major peak showed a single band (about 32 KD) when the gel was stained by Coomassie blue for proteins and periodic acid-Schiff reagent for glycoproteins after SDS-polyacrylamide gel electrophoresis. These findings suggest that the isolated and purified protein in the present study is P0 glycoprotein and the concentrated sample could be directly used as the antigen for preparing monoclonal antibody against PO glycoprotein.