PURIFICATION AND CHARACTERIZATION OF THE CA-125 TUMOR-ASSOCIATED ANTIGEN FROM HUMAN ASCITES
PURIFICATION AND CHARACTERIZATION OF THE CA-125 TUMOR-ASSOCIATED ANTIGEN FROM HUMAN ASCITES
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DOI：
10.1159/000217821
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发表时间：
1993-01-01
期刊：
影响因子：
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通讯作者：
WILDT, L
中科院分区：
文献类型：
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作者：
DELOSFRAILES, MT;STARK, S;WILDT, L
CA125是一种与上皮性卵巢癌相关的抗原决定簇，可被单克隆抗体OC125识别。CA125的生化结构、免疫学特性和生理功能尚不清楚，主要是因为表达它的分子尚未纯化至均一。在本研究中，我们开发了一种通过柱亲和层析纯化CA125的单一、一步法，使用OC125抗体作为固定配体。该柱对从人腹水（HA）中纯化CA125具有高度特异性。从柱上洗脱的抗原具有6240 ± 120单位CA125/毫克蛋白质的比活性，而初始HA样本中的比活性为100 ± 12单位/毫克蛋白质。纯化的免疫反应性CA125（IR - CA125）被证明本质上是蛋白质。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）和凝胶过滤特性表明，纯化的抗原以高分子量（MW）复合物形式存在，高达150万道尔顿，在强变性条件下可解离，产生表观分子量为205和55千道尔顿的部分。IR - CA125还与一种表观分子量为10 - 15千道尔顿的较低分子量蛋白质相关。通过从聚丙烯酰胺凝胶电洗脱后进行免疫放射测定，205千道尔顿分子量的蛋白质是免疫反应性CA125。此外，当亲和纯化的抗原进行SDS - PAGE，随后进行免疫印迹时，与碘化OC125抗体反应的泳道产生一条分子量为205千道尔顿的条带。我们的结果表明，在分析规模上，亲和柱对CA125的纯化是有用的。纯化的抗原正用于在体外研究中探究CA125在人卵巢癌的生长、发展和生理特性中的可能作用。
CA 125 is an antigenic determinant associated with epithelial ovarian carcinomas, which- is recognized by a monoclonal antibody, OC 125. The biochemical structure, the immunological characteristics and the physiological function of CA 125 are unknown, principally because the molecule expressing it has not been purified to homogeneity. In the present study, we developed a single, one-step method for purifying CA 125 by column affinity chromatography, using the OC 125 antibody as immobilized ligand. The column proved to be highly specific for the purification of CA 125 from human ascites (HA). The antigen that eluted from the column has a specific activity of 6,240 +/- 120 U of CA 125/mg protein, the specific activity in the initial HA samples being 100 +/- 12 U/mg protein. The purified, immunoreactive CA 125 (IR-CA 125) was shown to be proteinaceous in nature. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration characterization showed that the purified antigen exists as a high molecular weight (MW) complex, of up to 1. 5 million daltons, which could be dissociated under strong denaturing conditions, giving rise to moieties with an apparent MW of 205 and 55 kD. IR-CA 125 was also associated with a lower MW protein, with an apparent MW of 10-15 kD. The 205-kd MW protein was immunoreactive CA 125, as measured by immunoradiometric assay after being electroeluted from the polyacrylamide gel. Furthermore, when the affinity-purified antigen was subjected to SDS-PAGE, followed by immunoblotting, the lane which was reactive with the iodinated OC 125 antibody gave rise to a band with a molecular mass of 205 kD. Our results suggest that, on an analytical scale, the affinity column is useful for the purification of CA 125. The purified antigen is being used to investigate the possible role of CA 125 in the growth, development and physiological characteristics of human ovarian carcinomas in in vitro studies.