Neuropathological Diagnosis of Pulp Disease Using Antimyelin-protein Monoclonal Antibody

P0蛋白是一种完整的膜糖蛋白，占哺乳动物周围神经髓磷脂蛋白总量的50-60%。因此，在牙髓疾病的神经病理检查中，该糖蛋白可作为标记物。本研究旨在从一个小材料中分离和纯化P0糖蛋白。采用蔗糖密度梯度离心法制备大鼠腋窝神经、肌皮神经、正中神经、桡神经、尺神经和坐骨神经的髓磷脂组分。电镜观察髓磷脂组分。髓磷脂部分主要由片层结构组成。亚细胞细胞器，如细胞核和线粒体，在这部分中未观察到。髓磷脂组分在0.03 M HC1中匀浆，在4‰C下搅拌，离心。酸不溶性残留物直接用氯仿/再乙醇均质。将无脂沉淀物溶解在含有10%十二烷基硫酸钠（SDS）的0.1 M磷酸盐缓冲液中。将上清液涂于Sephacryl S-100色谱柱上，用含有2% SDS的0.1 M磷酸盐缓冲液平衡和洗脱。检测到一个主要的峰值。对主峰提取的馏分进行浓缩。sds -聚丙烯酰胺凝胶电泳后，蛋白质用考马斯蓝染色，糖蛋白用周期性酸-希夫试剂染色，主峰呈单条带（约32 KD）。以上结果表明，本研究分离纯化的蛋白为P0糖蛋白，浓缩后的样品可直接作为制备抗PO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.