Thingstad T, Haavik S, Hansen K, Sletten K, Codington J F, Barsett H
Department of Pharmacognosy, Institute of Pharmacy, P.O. Box 1068, Blindern, N-0316 Oslo, Norway.
Eur J Pharm Sci. 1998 Apr;6(2):121-9. doi: 10.1016/s0928-0987(97)00076-6.
Human carcinoma-associated antigen (HCA), detected by the mouse monoclonal anti-epiglycanin antibody, AE-3, has been isolated from ascitic fluid taken from a patient with metastatic ovarian adenocarcinoma and from spent medium of the human endometrial carcinoma cell line KLE-1 and compared with epiglycanin. The ascitic fluid and the spent medium were concentrated by a Filtron Ultrasette 100 K Omega membrane and fractionated by gel filtration on Sepharose CL-2B. The active fractions which consisted mainly of glycoproteins having relative molecular weights in the range 1000-2000 kDa compared to dextrans, were further purified by affinity chromatography on a column of immobilized AE-3. The active fraction was subjected to SDS-PAGE and blotted onto a PVDF membrane. The amino acid composition of HCA isolated from the two sources, were related but not identical and both showed some differences from the amino acid composition of epiglycanin. They all have, however, compositions typical of mucin-type glycoproteins. The isoelectric point for HCA from both KLE-1 and ascitic fluid were determined to be at pH 1.8 and the buoyant densities were about 1.4 g/ml as determined by cesium trifluoroacetate gradient centrifugation.
通过小鼠单克隆抗表膜糖蛋白抗体AE-3检测到的人癌相关抗原（HCA），已从一名转移性卵巢腺癌患者的腹水中以及人子宫内膜癌细胞系KLE-1的废弃培养基中分离出来，并与表膜糖蛋白进行了比较。腹水和废弃培养基通过Filtron Ultrasette 100 K Omega膜进行浓缩，并在Sepharose CL-2B上通过凝胶过滤进行分级分离。与葡聚糖相比，主要由相对分子质量在1000 - 2000 kDa范围内的糖蛋白组成的活性级分，通过固定化AE-3柱上的亲和色谱进一步纯化。活性级分进行SDS-PAGE并印迹到PVDF膜上。从这两种来源分离的HCA的氨基酸组成相关但不相同，并且与表膜糖蛋白的氨基酸组成都存在一些差异。然而，它们都具有粘蛋白型糖蛋白的典型组成。通过三氟乙酸铯梯度离心法测定，来自KLE-1和腹水的HCA的等电点均为pH 1.8，浮力密度约为1.4 g/ml。
