摘要
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2.59μg.L-1,灵敏度为0.75μg.L-1,检测限为1.0μg.L-1。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-1 and 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 °C 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.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2011年第6期857-864,共8页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
"十一五"国家科技支撑计划重大项目(2006BAK02A21)
作者简介
作者简介：王爱萍（1970-），女，青海西宁人，副教授，硕士生导师，主要从事动物免疫学研究，Tell：0371-65728928；E-mail：pingaw@126．com
通讯作者：李发弟，E-mail：lifd@gsau．edu．cn．
