雌二醇单克隆抗体制备及胶体金侧流层析免疫分析方法的建立

Preparation of monoclonal antibodies against 17β-estradiol and establishment of colloidal gold-based flow immunoassay

中文关键词:  雌二醇  单克隆抗体  间接竞争酶联免疫吸附实验  胶体金侧流层析免疫分析

英文关键词:17β-estradiol  monoclonal antibody  indirect competitive enzyme-linked immunosorbent assay  colloidal gold-based lateral flow immunoassay

中文摘要:

      目的 探讨雌二醇(17β-estradiol, E2)的3号位和17号位引入活性基团制备的半抗原对抗体灵敏度的影响, 选用灵敏度较高的抗体建立一种牛奶中E2胶体金侧流层析免疫分析方法(colloidal gold-based lateral flow immunoassay, CG-LFA)。方法 从E2的3号位羟基和17号位羟基引入活性基团分别制备半抗原H1a和H2, 偶联载体蛋白制备完全抗原, 经过动物免疫和细胞融合筛选, 制备单克隆抗体(monoclonal antibodies, mAbs), 采用间接竞争酶联免疫吸附实验(indirect competitive enzyme-linked immunosorbent assay, icELISA)对mAbs性能进行评估, 筛选出灵敏度最高的mAb, 最后采用静电吸附法将胶体金标记抗体为探针, 构建牛奶中E2的CG-LFA。结果 制备了H1a-7B7、H1a-7B12、H1a-9C7、H1a-10E7、H2-2E2、H2-3C10、H2-4A10和H2-6D2 8种mAbs, 经过icELISA评估, 其半数抑制浓度(half maximal inhibitory concentration, IC50)分别为0.116、0.207、0.072、0.370、0.442、6.170、0.415和4.411 ng/mL, 最终选取H1a-9C7, 构建牛奶中E2的LFA, 消线(cut-off)值为6.00 ng/mL, 与苯甲酸雌二醇和雌三醇的交叉反应率分别为101.31%和2.43%, 在真实样本加标回收实验中, 检测结果与液相色谱-串联质谱法(liquid chromatography-tandem mass spectrometry, LC-MS/MS)结果一致。结论 E2的3号位引入活性基团设计合成的半抗原所制备的E2抗体灵敏度, 比17号位引入活性基团制备的抗体灵敏度高, 并且建立的LFA准确度良好, 为牛奶中E2快速检测提供了一定的技术手段。

英文摘要:

      Objective To investigate the impact of immunogens derived from site 3 and site 17 of 17β-estradiol (E2) on antibody sensitivity, and establish a colloidal gold-based lateral flow immunoassay (CG-LFA) for the quantitative analysis of E2 in milk. Methods The haptens of H1a and H2 were prepared by introducing active groups from the site 3 and site 17 hydroxyl groups of E2, and then they were coupled with carrier protein to prepare complete antigens. After animal immunization and cell fusion screening, the monoclonal antibodies (mAbs) were prepared. The performance of mAbs were evaluated by indirect competitive enzyme-linked immunosorbent assay (icELISA), and the most sensitive mAbs were selected. Finally, CG labeled mAbs were employed as probes using electrostatic adsorption method to construct CG-LFA for the detection of E2 in milk. Results Eight kinds of mAbs, named H1a-7B7, H1a-7B12, H1a-9C7, H1a-10E7, H2-2E2, H2-3C10, H2-4A10 and H2-6D2, were prepared. After evaluation by icELISA, half maximal inhibitory concentration (IC50) were 0.116, 0.207, 0.072, 0.370, 0.442, 6.170, 0.415 and 4.411 ng/mL, respectively. Finally, H1a-9C7 was selected to construct the LFA of E2 in milk. Cut-off value of the CG-LFA was 6.00 ng/mL, and the cross-reactivity with β-estradiol and 3-benzoateand estriol were 101.31% and 2.43%, respectively. In the real sample recovery experiment, the results were consistent with those of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Conclusion The sensitivity of E2 antibodies prepared from haptens designed with the introduction of the site 3 active group of E2 is higher than that of the site 17. The established LFA demonstrates high accuracy, which provides a certain technical solution for the rapid detection of E2 in milk.