检测动物组织中金刚烷胺残留的酶联免疫试剂盒的研制

Development of an enzyme-linked immunosorbent assay kit for the determination of the amantadine residues in animal tissues

通过对金刚烷胺分子结构进行改造,制备得到了金刚烷胺半抗原及人工抗原,免疫动物,制备特异性单克隆抗体。在筛选金刚烷胺单克隆抗体的基础上,建立酶联免疫检测方法,并研制出检测动物组织中金刚烷胺残留的试剂盒。该试剂盒工作范围为0.5～40.5μg/L,线性回归方程为y=-2.069x＋0.493,IC_（50）为2.1μg/L,相关系数R2为0.998;试剂盒检测限为0.25μg/kg,金刚烷胺回收率在82.5%～91.0%,批内、批间变异系数均小于10%;与阿莫西林、苯唑西林、头孢噻呋三种类似结构药物均无交叉反应。该试剂盒灵敏度高、检测限低、特异性强、操作简便,可广泛用于动物组织中金刚烷胺残留量的测定。

The aim of this study is to prepare an enzyme-linked immunosorbent assay kit for the qualitative or half-quantitative determination of amantadine residues in animal tissues. Amantadine hapten was obtained by chemical synthesis. Immunogen and the coated antigen were prepared by amantadine hapten coupled to carrier protein BSA and OVA,respectively,and monoclonal antibody was prepared. Then the direct competitive ELISA method was used to develop a detection kit,and the technical parameters of the kit was evaluated. Results showed that amantadine was successfully coupled with BSA and OVA,respectively,by TNBS. The working range of amantadine kit was 0. 5-40. 5μg /L,the obtained regression equation was y =-2. 069x＋ 0. 493,the IC_（50） was 2. 1 μg / L,and the R2 was 0. 998. The limit of detection（ LOD） of ELISA kit was 0. 25 μg/kg,the recovery was in a range of 82. 5% and 91. 0%,and inter-and intra-coefficients of variation（ CVs） was less than 10%. There was no cross-reactivity towards drugs with similar structure,such as amoxicillin,oxacillin and ceftiofur. It suggests that amantadine ELISA kit established in this study has high sensitivity,low detection limit,strong specificity and simple operation,and can be widely used in the determination of amantadine residue in animal tissues.