Nodal检测双单抗夹心ELISA法建立及应用  

Development and Application of A Double Monoclonal Antibody Sandwich ELISA for the Assay of Nodal

作　　者：吴孟玲 周嘉旺 杜军[1] 

机构地区：[1]中山大学药学院微生物与生化药学实验室,广州510006

出　　处：《中国生物工程杂志》2017年第3期51-57,共7页China Biotechnology

基　　金：国家自然科学基金资助项目(81672943;81472643)

摘　　要：现已证实,Nodal参与了肿瘤恶性生物学过程,但对其高敏感检测法尚未建立。采用基因工程表达人源Nodal作为抗原免疫BALB/c小鼠,通过经典PEG诱导的细胞融合技术筛选出针对Nodal的特异性单克隆抗体7株。夹心ELISA确证7株抗体组成了15种可配对的抗体对。经筛选后选取抗体对AF12-DG5建立标准化夹心ELISA法,结合生物素-亲和素检测系统,DG5抗体标记生物素,采用链亲和素与辣根过氧化物酶标记的生物素(HRP-Biotin)按质量比4∶1预先混合孵育的ABC混合物进行检测,以提高ELISA法的灵敏度。棋盘滴定确定抗体工作最佳浓度为:捕获抗体(AF12)2μg/ml,检测抗体(生物素化DG5)2μg/ml。此条件下的夹心ELISA法线性范围为0~3 000pg/ml,检测限为68pg/ml,平均回收率为99.6%,精密度准确度良好。以正常人血清作为阴性对照,使用该夹心ELISA法测定结直肠癌、鼻咽癌和胆囊癌患者血清,发现三种肿瘤患者血清与正常人血清中的Nodal浓度均存在明显的统计学差异,可作为临床使用参考。Background： Nodal is a member of the transforming growth factor beta（ TGF-beta） super family,essential in maintaining the pluripotency of human embryonic stem cells（ h ESCs）,which is not active in most adult tissues,its reexpression and signaling have been linked to multiple types of human cancer. Recent findings have revealed that Nodal is a critical regulator of melanoma growth,plasticity and tumorigenicity,and holds promise as a new tumor biomarker. Similar observations have been reported in multiple human cancers. To utilize Nodal as a useful biomarker for cancer detection,a double monoclonal antibody sandwich enzyme-linked immunosorbent assay（ ELISA） may be a reliable,easy and cheap means. Methodology/principal findings：Recombinant Nodal was used as antigen to immunize BALB/c mice,by cell fusion between plasma cells and SP2/0 cells,7 specific positive monoclonal antibodies were picked out,which were verified through indirect ELISA,Western blot and flow cytometry analyses. Therefore 15 pairs of antibodies can be used to set up a double monoclonal antibody sandwich ELISA,AF12-DG5 was the most appropriate antibody pairs. Combined with biotin-avidin systerm,DG5-biotin was used as detection antibody while the complex of streptavidin and HRPbiotin（ mass ratio 4 ∶ 1） as detection reagent. The best working concentration of antibodies were AF12 2μg/ml and DG5-biotin 2μg/ml. Linear range of this ELISA is 0 ～ 3 000pg/ml,regression equation is y = 0. 000 3x ＋0. 150 5,R2= 0. 999,low limit of detection is 68pg/ml,average recovery is 99. 6%. The result of serum detection indicates that more Nodal was expressed in 70% of gallbladder carcinoma patients, 92% of nasopharyngeal carcinoma patients and 97% of colorectal carcinoma patients, which were statistically significantly increased compare to normal human. Conclusions/significance： A sensitive and specific Nodal ELISA test has been developed. This test is high sensitive and easy to use and can discriminate cancer patients from normal person spe

关 键 词：双抗夹心ELISA NODAL 细胞融合 配对抗体 生物素-亲和素 

分 类 号：Q816[生物学—生物工程]