本研究旨在建立新霉素(Neomycin,NEO)免疫学检测试剂盒.以制备的抗NEO单克隆抗体(NEO mAb)为基础,建立阻断ELISA分析方法,组装新霉素阻断ELISA试剂盒,并对试剂盒的性能进行鉴定.结果表明,NEO试剂盒(NEO-kit)标准曲线呈典型的S型,符合4参数logit曲线拟合,相关系数R2=0.990 3;检测范围为1.0～64.0μg·L-1,半数抑制浓度(IC50)为2.59 μg·L-1,灵敏度为0.75 μg·L-1,检测限为1.0 μg·L1.试剂盒除了和NEO阳性样品反应呈阳性外,与庆大霉素、链霉素、土霉素、沙丁胺醇、环丙沙星、二氟沙星、磺胺嘧啶及磺胺甲噁唑等其他药物均无交叉反应性.奶样、饲料样及肉样的平均添加回收率为89.50%、89.58%和87.92%;平均批内和批间变异系数均小于15%,而且批间变异系数小于批内变异系数;基质对NEO-Kit的检测结果影响比较小;NEO-Kit在4℃可保存6个月以上.试剂盒和HPLC-MS-MS对牛奶中新霉素回收率没有显著差异(P≥0.05).试剂盒和HPLC-MS-MS对饲料样检测结果也无显著差异(P≥0.05).本研究成功研制出敏感、特异、准确的NEO检测试剂盒.%A blocking ELISA kit for detection of neomycin was developed based on the NEO mAb, and its properties were examined. The result showed that the calibration curve of NEO-kit was a typical sigmoid curve fitted to the four parameters logistic equation with the linear detection of 1.0 to 64.0 μg · L-1 and R2 was 0. 990 3. The kit has a sensitivity of 0. 75 μg · L-1 , the IC50 of 2. 59 μg · L-1and the detection limit of 1. 0 μg · L-1. The kit had no cross-reactivity with gentamicin, streptomycin, oxytetracycline, ciprofloxacin, difloxacin, salbutamol, sulfadiazine and sulfamethoxazole. The recoveries of NEO in milk, feed and meat were 89. 50％, 89.58％ and 87. 92％, respectively. The inter-assay and intra-assay coefficient variation were all below 15％.Moreover, the inter-assay coefficient variation was lower than that of intra-assay. Different sample diluted solution had very little effect on the NEO-kit. The kit remained effective for storing at 4 ℃ more than six month. There was no significant difference of the recovery of neomycin in milk between with NEO-kit and with HPLC-MS-MS (P≥0. 05). The neomycin content in feed determined by NEO-kit and HPLC-MS-MS has no significant difference ( P≥0. 05). In conclusion, we developed a NEO-kit with high sensitivity, specificity and accuracy.
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