摘要
本论文利用胶体金作为标记物,根据竞争抑制免疫层析原理,对磺胺类兽药残留快速检测试纸条方法进行了系统的研究。通过优化各项实验指标,利用磺胺类药物多克隆抗体和半抗原偶联物(S_4-OVA)研制出磺胺类多残留免疫层析试纸条;利用抗磺胺二甲嘧啶多克隆抗体和半抗原偶联物(SMZ-OVA)研制出磺胺二甲嘧啶免疫层析试纸条。
采用柠檬酸三钠法制备40 nm胶体金,电镜鉴定后标记多克隆抗体,半抗原-OVA和羊抗兔二抗分别作为检测带和质控带包被在硝酸纤维素膜上,金标记抗体喷涂在玻璃纤维垫上,制成免疫层析快速检测试纸条。
磺胺二甲嘧啶试纸条的方法检出限为1μg/L,磺胺类多残留试纸条方法检出限:磺胺噻唑、磺胺异噁唑、磺胺对甲氧嘧啶、磺胺甲氧嗪、磺胺吡啶分别为2μg/L、2μg/L、5μg/L、5μg/L、5μg/L。
以猪肉、鸡肉、牛奶、鲫鱼为实际样品(经HPLC检测为阴性样品)进行磺胺类药物的添加回收实验,以甲醇为提取溶剂,磷酸盐缓冲液PBS-0.05%Tween为基质掩蔽剂,稀释一定倍数后即可消除基质影响,用于试纸条的检测。五种磺胺药物在四种样品基质中的最低检出限均等于或低于MRL(100μg/kg);磺胺二甲基嘧啶在四种样品基质中的最低检出限均等于或低于MRL(20μg/kg)。
通过高效液相色谱法验证了试纸条检测方法的有效性。结果表明试纸条在其检测范围内与HPLC检测结果一致性较好,验证了该检测方法的有效性。
试纸条分别密封保存于4℃、37℃一段时间后做稳定性测试,结果表明在4℃密封干燥条件下试纸条放置6个月内可保持检测结果的可靠性。
该试纸条检测方法灵敏度高,快速,简便,10 min内即可得出检测结果。所研制的两种试纸条适用于动物源性食品中磺胺类药物残留的大批量现场检测。
A competitive assay based on the principle of immunoassay using colloidal gold as tracer for the rapid on-site detection of sulfonamides residues in food samples was studied. After optimizing the various factors and conditions of the test, using the anti-sulfonamides polyclone antibody, S_4-OVA, the anti-SMZ antibody and SMZ-OVA, the colloidal gold immunochromatographic rapid test strip was developed for the determination of sulfonamides residues.
Gold chloride was reduced with sodium citrate to make 40 nm colloidal gold, then it was labeled to the polyclonal antibody after identification. The hapten-OVA and goat anti-rabbit IgG were dispense on test line and control line respectively and the polyclonal antibody was solidified to glass fibers to prepare immunochromatographic strips.
The limit of detection of SMZ test strip was 1 ug/L, the limits of detection of sulfonamides (STZ, SIZ, SME, SMPD, SP) were 2,2, 5, 5, 5μg/L.
Pig muscle, chicken muscle, milk and fish muscle samples were used as real sample (determined to be free of sulfonamides by HPLC) for analysis. These food samples were spiked with sulfonamides. For the one-step strip test, the methanol was used as the extract solvent, and the matrix effect could be removed by dilution with PBST. The LOD of five sulfonamides in four samples were equal to or lower than maximum residue limit (MRL, 100μg/kg). The LOD of SMZ in four samples was equal to or lower than maximum residue limit (MRL, 20μg/kg).
The established method was validated by the instrumental method of HPLC. The results obtained by test strip were coherence with the results obtained by HPLC. The results indicated that the test strips can be applied in the rapid detection of sulfonamides residues.
The immunochromatographic rapid test strips were placed at 4℃and 37℃for several days and then were used to detect the sulfonamides.The stability test indicated that the reliability of test strips were good at 4℃within 6 months.
The method established is sensitive, rapid and simple, and the results could be obtained within 10 minutes. All the results proved that the two test strips could be widely used to detect the sulfonamides residues in animal foods on-site.
引文
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