该研究建立一种基于免疫磁珠分离、净化和富集的快速、灵敏的胶体金免疫层析同时检测猪肉中的3-甲基-喹噁啉-2-羧酸(3-methylquinoxaline-2-carboxylic acid,MQCA)和喹噁啉-2-羧酸(quinoxaline-2-carboxylic acid,QCA)残留的方法。利用免疫磁珠快速提取猪肉中的MQCA和QCA,磁场快速分离,加热洗脱并富集,然后用胶体金免疫层析法检测,可在5 min内完成,MQCA定性和定量检测限均为0.1 μg/kg(QCA定性和定量检测限均为0.25 μg/kg)。结果表明,经过优化后试纸条最佳工艺为抗体标记浓度2.5 mg/mL,抗原(T线)划膜浓度1.2 mg/mL,羊抗鼠二抗(C线)划膜浓度1.0 mg/mL,制备免疫磁珠时亲和素磁珠与抗体最佳偶联量为40 μg/mg,MQCA和QCA样本添加浓度分别为0.1、0.5、2和0.25、1.25、5 μg/kg,回收率为89.9%~115.2%,变异系数为6.8%~11.5%。用基于免疫磁珠的胶体金免疫层析法和液相色谱-质谱/质谱技术(liquid chromatography-tandem mass spectrometry, LC-MS/MS)同时对20个市售猪肉样本检测,一致性较好,证明胶体金免疫检测方法可以用于实际猪肉样本中MQCA和QCA的快速定性和定量检测。
A sensitive colloidal gold immunochromatographic assay (CGICA) was established for simultaneous determination of 3-methyl-quinoxaline-2-carboxylic acid (MQCA) and quinoxaline-2-carboxylic acid (QCA) in pork. The method is using immunomagnetic beads to separate, purify and enrichment MQCA and QCA. The qualitative and quantitative determination cloud be finished within 5 min. The limit of detection (LOD) of qualitative and quantitative detection is 0.1 μg/kg (0.25 μg/kg for QCA). The optimized results showed that concentration of monoclonal antibody (anti-MQCA), antigen (Test line) and goat anti-mouse IgG (Control line) were 2.5, 1.2 and 1.0 mg/mL respectively. The optimized capture monoclonal antibody (anti-MQCA) for immunomagnetic beads was 40 μg/mg. When MQCA was spiked in pork at the concentration of 0.1, 0.5 and 2 μg/kg, QCA was spiked in pork at the concentration of 0.25, 1.25, 5 μg/kg; the recovery range was from 89.9% to 115.2%, and coefficient of variation was from 6.8% to 11.5%. Furthermore, 20 samples were analyzed with the developed CGICA, and results were correlated well with those obtained in liquid chromatography-tandem mass spectrometry (LC-MS/MS). This shows the potential of using CGICA for determination of MQCA and QCA in pork.
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