卵巢上皮性癌单克隆抗体183B2相关抗原的纯化和鉴定

Purification and characterization of corresponding antigen recognized by monoclonal antibody 183B2 against ovarian carcinoma

目的纯化卵巢上皮性癌单克隆抗体(单抗)183B2相关抗原,并对该抗原及其理化特性进行鉴定和分析。方法用间接酶联免疫吸附试验(ELISA)方法筛选出与单抗183B2阳性反应较强的卵巢上皮性癌腹水标本,继而用免疫亲和层析技术纯化得到相应抗原。纯化后的抗原分别经高碘酸钠、神经氨酸酶、去糖脂混合液及胰蛋白酶、链酶蛋白酶破坏糖链和肽链后,用间接ELISA方法检测其活性,并用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及蛋白印迹(western blot)法进行鉴定。再进一步用双向电泳结合western blot对卵巢上皮性癌细胞株SKOV3细胞的相应抗原进行验证。结果纯化后的抗原经破坏糖链处理后活性不变,而经破坏肽链及加热处理后活性消失。SDS-PAGE显示,该抗原两个不同亚单位的相对分子质量分别为56000和25000,而前者能与单抗183B2反应。纯化后的抗原N端氨基酸测序后进行同源序列分析发现,此序列与人Ig重链N端的15个氨基酸相同。确定了双向电泳凝胶上3个目标点的位置,并初步确定该蛋白点的相对分子质量约为56000,等电点为5.3～5.8。结论卵巢上皮性癌单抗183B2相关抗原决定簇位于糖蛋白的肽链上,为Ig超家族成员,它可能是一种新的肿瘤相关抗原。

Objective To purify MAb183B2 corresponding antigen of ovarian carcinomas and study its physical and chemical characteristics. Methods The strongest positive samples reacted with MAb183B2 were screened from ovarian cancer ascites with indirect enzyme linked immunosorbent assay (ELISA). Then the antigen was purified from ascitic fluid by affinity chromatography with MAb183B2. Nature of the purified antigen was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot and its reactivity was determined after treatment with NaIO4, trypsin, pronase, neuraminidase, deglycolipid mixture and heating. Two dimensional electrophoresis combined with western blot was then used to isolate and identify the antigen from ovarian cancer cell line SKOV3. Results The reactivity of the antigen after treated by NaIO4, neuraminidase and deglycolipid mixture was still positive, while it was negative after treated by trypsin, pronase and heating. The antigen proved to be composed of two different subunits of 56 000 and 25 000 by SDS-PAGE, and MAb183B2 could blot the 56 000 band by western blot. The antigen from the SKOV3 cell line displayed microheterogeneity appearing as three spots over a pI range 5.3～5.8 at 56 000. Conclusions The epitope of 183B2-antigen is at its peptide core. The antigen belongs to the Ig super family and might be a new ovarian carcinoma associated antigen.