Rojekittikhun W, Yamashita T, Saito S, Watanabe T, Azuma T, Sendo F
Department of Immunology and Parasitology, Yamagata University School of Medicine, Japan.
Southeast Asian J Trop Med Public Health. 1993 Dec;24(4):680-4.
An immunoaffinity column was prepared by coupling a partially purified Gnathostoma spinigerum-specific IgG1, MAb SK-6C4 (5 mg/ml) to CNBr-activated Sepharose 4B. Ten milliliters of approximately 0.3 mg/ml of crude soluble G. spinigerum larval antigens (GsAL3) were loaded onto the affinity column at a flow rate of about 5 ml/hour. Elution of the bound antigens was accomplished using 50 mM diethylamine-HCI containing 0.15 M NaCL, pH 11.5. The average amount of eluted antigens obtained by one passage of crude GsAL3 (1-4 mg) through 4 to 8 ml of column matrix was 143 micrograms (range, 67 - 414 micrograms). The minimal amount of purified GsAL3 detectable by ELISA using MAb SK - 6C4 (100 micrograms/ml) was 50 ng/ml. The SK - 6C4 affinity-purified GsAL3 was found to be relatively pure and immunologically specific as determined by SDS-PAGE and Western blotting, respectively.
通过将部分纯化的棘颚口线虫特异性IgG1单克隆抗体SK-6C4（5毫克/毫升）偶联到溴化氰活化的琼脂糖凝胶4B上，制备了免疫亲和柱。将10毫升约0.3毫克/毫升的棘颚口线虫幼虫粗可溶性抗原（GsAL3）以约5毫升/小时的流速加载到亲和柱上。使用含有0.15摩尔/升氯化钠、pH值为11.5的50毫摩尔二乙胺-盐酸洗脱结合的抗原。通过4至8毫升柱基质对粗GsAL3（1至4毫克）进行一次过柱所获得的洗脱抗原平均量为143微克（范围为67至414微克）。使用单克隆抗体SK-6C4（100微克/毫升）通过酶联免疫吸附测定法可检测到的纯化GsAL3的最小量为50纳克/毫升。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和蛋白质印迹法分别测定发现，SK-6C4亲和纯化的GsAL3相对纯净且具有免疫特异性。
