摘要
采用对碘苯酚增强的HRP-鲁米诺-H2O2化学发光体系,建立了虾肉中呋喃它酮代谢物5-吗啉甲基-3-氨基-2-恶唑烷基酮(AMOZ)残留的间接竞争化学发光酶免疫分析(icCLEIA)检测方法。检测所用抗体是基因重组的抗AMOZ衍生物单链抗体。优化的icCLEIA最佳工作条件为:AMOZA-OVA包被浓度62.5μg/L,抗AMOZ衍生物单链抗体最佳稀释度为1∶10,竞争免疫反应时间45 min,HRP酶标记羊抗鼠抗体最佳稀释度为1∶10000,孵育时间50 min。本方法的IC50为1.38μg/L;灵敏度为0.09μg/L;线性范围为0.26～9.08μg/L(IC20～IC80);批内和批间相对标准偏差均小于15%;抗AMOZ衍生物单链抗体与其它硝基呋喃类抗生素及其代谢物均没有交叉反应,特异性良好。4个不同添加量的AMOZ加标样品的平均回收率分别为72.2%,73.4%,72.6%和78.6%。与HPLC-MS/MS法测定值进行比较发现,两种方法相关性良好(R2=0.9997)。本方法可用于水产品中AMOZ残留的快速检测。
An indirect competitive chemiluminescence enzyme immonoassay（icCLEIA） for the quantitative analysis of furaltadone metabolite 5-morpholinomethyl-3-amino-2-oxazolidone（AMOZ） in shrimp sample was established with the highly sensitive luminol-H2O2-horseradish peroxidase（HRP）-4-iodophenol chemiluminescence reaction detection system.The icCLEIA method was based on a single-chain variable fragment（scFv） antibody against AMOZ derivative.The optimized assay conditions were 62.5 μg/L of coating antigen（5-morpholinomethyl-3-（glyoxalamino）-2-oxazolidone-ovalbumin,AMOZA-OVA）,the dilute time of scFv antibody was 1 ∶ 10,the immunoreaction time was 45 min,the dilute times and incubation time of HRP-goat anti mouse IgG were 1 ∶ 10000 and 50 min,respectively.The icCLEIA results showed IC50 value,limit of detection（LOD） and linear range were 1.38 μg/L,0.09 μg/L and 0.26-9.08 μg/L,respectively.Inter-assay and intra-assay RSD was both below 15%.The scFv antibody showed high specificity.The average recoveries of four spiked level of AMOZ were 72.3%,73.4%,72.6% and 78.6%,respectively.Compared with HPLC-MS/MS,there was a good correlation between the two methods（R2=0.9997）.Thus,the established icCLEIA method could be further used for detecting AMOZ in aquatic products samples rapidly and efficiently.
出处
《分析化学》
SCIE
EI
CAS
CSCD
北大核心
2012年第12期1816-1821,共6页
Chinese Journal of Analytical Chemistry
基金
国家自然科学基金(No.30871755)
广东省科技计划项目(No.1211150100102)
广东省食品质量安全重点实验室(华南农业大学)开放基金(No.09003566)资助
关键词
呋喃它酮代谢物AMOZ
刀额新对虾
间接竞争化学发光酶免疫分析
辣根过氧化物酶
Furaltadone metabolite 5-morpholinomethyl-3-amino-oxazoldone
Metapenaeus ensis
Indirect competitive chemiluminescence enzyme immunoassay
Horseradish peroxidase
