间接竞争酶联免疫吸附法检测保健食品中糖皮质激素类药物

Determination of glucocorticoid drugs in health foods by indirect competitive enzyme-linked immunosorbent assay

目的建立间接竞争酶联免疫吸附(indirect competitive enzyme-linked immunosorbent assay,ic-ELISA)法用于保健食品中糖皮质激素类药物多残留检测。方法将可的松、地塞米松分别与丁二酸酐衍生制备免疫半抗原,倍他米松衍生制备包被半抗原,采用活泼酯法与载体蛋白偶联制备人工抗原,采用紫外(ultraviolet,UV)吸收光谱鉴定及基质辅助激光解析串联飞行时间质谱(matrix assisted laser desorption-tandem time of flight-mass spectrometry,MALDI-TOF-MS)测定人工抗原的偶联比。通过动物免疫,制备针对糖皮质激素类药物的兔多克隆抗体,建立保健食品中糖皮质激素类药物多残留检测ic-ELISA法。结果地塞米松丁二酸酐衍生物为免疫半抗原,倍他米松丁二酸酐衍生物为包被半抗原。通过优化实验条件,确定了最佳的包被原质量浓度为1.25μg/m L,以及最佳的抗体稀释倍数为9000倍。在此条件下,本研究建立的ic-ELISA法对11种糖皮质激素类药物的半抑制质量浓度为0.68~40.17 ng/m L,展现了良好的检测灵敏度。使用甲醇超声提取,样品提取液通过标准稀释液稀释500倍消除基质效应,11种目标分析物在4种剂型保健食品中的添加回收率为81.2%~118.7%,变异系数低于14.00%。结论本研究成功制备糖皮质激素类药物的广谱性多克隆抗体,并建立了保健食品中糖皮质激素类药物多残留检测ic-ELISA法,可用于保健食品中糖皮质激素类药物的快速筛查。

Objective To establish an indirect competitive enzyme-linked immunosorbent assay(ic-ELISA)for multi residue detection of glucocorticoid drugs in health foods.Methods Cortisone and dexamethasone were respectively derived with succinic anhydride to prepare immunizing half-antigens,while betamethasone was derived to prepare encapsulated half-antigens.Artificial antigens were prepared by coupling with carrier proteins using the active ester method.The coupling ratio of the artificial antigens was determined by ultraviolet absorption spectroscopy and matrix assisted laser desorption tandem time-of-flight mass spectrometry(MALDI-TOF-MS).Through animal immunity,prepare rabbit polyclonal antibodies targeting glucocorticoid drugs,and establish an ELISA method for multi residue detection of glucocorticoid drugs in health foods.Results Dexamethasone succinic anhydride derivatives were immunizing half-antigens and betamethasone succinic anhydride derivatives were encapsulated half-antigens.By optimizing the experimental conditions,the optimal mass concentration of the encapsulant was determined to be 1.25μg/mL,and the optimal antibody dilution was determined to be 9000-fold.Under these conditions,the semi-inhibitory concentration range of the ic-ELISA established in this study was 0.68–40.17 ng/mL for 11 kinds of glucocorticoid drugs,demonstrating good detection sensitivity.The methanol was extracted by ultrasonic extraction,and the sample extracts were diluted 500-fold by standard diluent to eliminate matrix effect,the recoveries of the 11 kinds of target analytes spiked in 4 kinds of dosage forms of health food products ranged from 81.2%–118.7%,with the coefficients of variation lower than 14.0%.Conclusion The wide-spectrum polyclonal antibodies for glucocorticoid drugs have been successfully prepared,and the ic-ELISA method for multi-residue detection of glucocorticoid drugs in health foods has been established,which can be used for rapid screening of glucocorticoid drugs in health foods.