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

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 specifically. The achieved data suggest that the developed ELISA may be applicable as a research tool for detection of human cancer.