Nodal检测双单抗夹心ELISA法建立及应用
发布时间：2019-03-14 13:18
【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。
[Abstract]:It has been proved that Nodal is involved in malignant biological process of tumor, but its high sensitivity detection has not been established. BALB/c mice were immunized with genetically engineered human Nodal as antigen. Seven specific monoclonal antibodies against Nodal were screened by classical PEG-induced cell fusion technique. Sandwich ELISA confirmed that 15 pairing antibody pairs were made up of 7 antibodies. After screening, the antibody was selected to set up a standardized sandwich ELISA method for AF12-DG5, combined with biotin-avidin detection system, and DG5 antibody was used to label biotin. In order to improve the sensitivity of ELISA method, the mixture of HRP-Biotin and HRP labeled biotin (ABC) was detected by pre-incubation with ABC mixture at a mass ratio of 4:1 in order to improve the sensitivity of the method. The optimal concentration of antibody determined by chessboard titration was as follows: capture antibody (AF12) 2 渭 g / ml, to detect antibody (biotin DG5) 2 渭 g / ml. Under these conditions, the linear range of sandwich ELISA method is 0 ~ 3 000 PG / ml, the detection limit is 68 PG / ml, the average recovery is 99.6%, and the accuracy of precision is good. The serum levels of Nodal in patients with colorectal cancer, nasopharyngeal carcinoma and gallbladder carcinoma were determined by sandwich ELISA method with normal human serum as negative control. The results showed that there were significant differences in serum Nodal concentrations between the three kinds of tumor patients and normal controls. It can be used as a reference for clinical use.
【作者单位】： 中山大学药学院微生物与生化药学实验室;
【基金】：国家自然科学基金资助项目(81672943、81472643)
【分类号】：R730.4
，
本文编号：2440016
[Abstract]:It has been proved that Nodal is involved in malignant biological process of tumor, but its high sensitivity detection has not been established. BALB/c mice were immunized with genetically engineered human Nodal as antigen. Seven specific monoclonal antibodies against Nodal were screened by classical PEG-induced cell fusion technique. Sandwich ELISA confirmed that 15 pairing antibody pairs were made up of 7 antibodies. After screening, the antibody was selected to set up a standardized sandwich ELISA method for AF12-DG5, combined with biotin-avidin detection system, and DG5 antibody was used to label biotin. In order to improve the sensitivity of ELISA method, the mixture of HRP-Biotin and HRP labeled biotin (ABC) was detected by pre-incubation with ABC mixture at a mass ratio of 4:1 in order to improve the sensitivity of the method. The optimal concentration of antibody determined by chessboard titration was as follows: capture antibody (AF12) 2 渭 g / ml, to detect antibody (biotin DG5) 2 渭 g / ml. Under these conditions, the linear range of sandwich ELISA method is 0 ~ 3 000 PG / ml, the detection limit is 68 PG / ml, the average recovery is 99.6%, and the accuracy of precision is good. The serum levels of Nodal in patients with colorectal cancer, nasopharyngeal carcinoma and gallbladder carcinoma were determined by sandwich ELISA method with normal human serum as negative control. The results showed that there were significant differences in serum Nodal concentrations between the three kinds of tumor patients and normal controls. It can be used as a reference for clinical use.
【作者单位】： 中山大学药学院微生物与生化药学实验室;
【基金】：国家自然科学基金资助项目(81672943、81472643)
【分类号】：R730.4
，
本文编号：2440016
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