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肿瘤细胞相关抗原对肿瘤临床诊断及预后监测具有重要意义.近年来应用单克隆抗体技术,鉴定出许多肿瘤相关抗原.目前在临床上应用的结直肠癌标志物除CEA外,还有CA19-9、结直肠癌相关抗原(CCA)等.本文采用鼠抗人结直肠癌单克隆抗体纯化结肠癌组织中的CCA,并对此抗原作初步分析.1 材料与方法1.1 可溶性抗原的制备 人结肠癌组织用生理盐水洗净.将肿瘤组织剪成小块,加入0.01mol/LpH7.2(含3mol/L氯化钾)磷酸缓冲液,制成匀浆.4℃搅拌14h.10000r/min离心30min,上清液用上述缓冲液透析48h.将此提取液边搅拌边滴加3mol/L 高氯酸,至其最终浓度成0.6mol/L,4℃搅拌30min,10000r/min离心30min,上清液透析,稍作浓缩.测定蛋白浓度为2.5×10~2mg/ml.1.2 单抗亲和层析柱的制备和亲和纯化CCA将抗CCA单克隆抗体腹水(IgGl,中国科学院细胞生物研究所提供)经二次盐析后,用pH8.3 0.1mol/L 碳酸缓冲液透析平衡.每毫升 CNBr活化Sepharose 4B凝胶加20mg抗体蛋白混合.室温倒转反应2h,继用1mol/L.pH7乙醇胺封闭2h,制成单抗亲和层析柱.最后用0.15mol/L pH7.2磷酸缓冲液平衡,装柱.将上述高氨酸提取液上亲和层析柱.解高用0.2mol/L pH2.5甘氨酸-HCI缓冲液,收集并及时中和所得抗原蛋白液.1.3 免疫酶法测定 取市售的CCA试剂盒(中科院及本
Tumor cell-associated antigens are important for clinical diagnosis and prognostic monitoring of tumors. In recent years, monoclonal antibody technology has been used to identify many tumor-associated antigens. In addition to CEA, CA19-9 is currently used as a marker of colorectal cancer in clinical applications. Colorectal cancer-associated antigen (CCA), etc. In this study, CCA in colon cancer tissues was purified using mouse anti-human colorectal cancer monoclonal antibody, and preliminary analysis was performed on this antigen. 1 Materials and methods 1.1 Preparation of soluble antigen Human colon cancer The tissue was washed with normal saline. The tumor tissue was cut into small pieces and 0.01 mol/L pH7.2 (containing 3 mol/L potassium chloride) phosphate buffer was added to prepare a homogenate. The mixture was centrifuged at 4°C for 14 h, 10000 rpm, and centrifuged for 30 min. The supernatant was dialyzed for 48 h with the above buffer. This extract was added dropwise with 3 mol/L perchloric acid to a final concentration of 0.6 mol/L, stirred at 4°C for 30 min, and centrifuged at 10000 r/min for 30 min. Liquid dialysis, slightly concentrated. Determination of protein concentration of 2.5 × 10 ~ 2mg/ml.1.2 Preparation of monoclonal antibody affinity column and affinity purification of CCA anti-CCA monoclonal antibody ascites (IgGl, Institute of Cell Biology, Chinese Academy of Sciences) ) After secondary salting out, equilibrate with pH 8.3 0.1 mol/L Carbonate buffer. Each milliliter of CNBr activated Sepharose 4B gel plus 20mg of antibody protein mixed. Inverted reaction at room temperature for 2h, followed by 1mol/L. pH7 ethanolamine blocked 2h, made a monoclonal antibody affinity chromatography column. Finally with 0.15mol/L pH7.2 Phosphate buffer solution was equilibrated and packed in the column. The above-mentioned high-acid extraction solution was applied to an affinity chromatography column. The solution was eluted with 0.2 mol/L pH2.5 glycine-HCI buffer and collected and timely neutralized to obtain the antigen protein solution. 1.3 Immunization Enzymatic determination of commercially available CCA kits (Chinese Academy of Sciences and Ben