摘要
以氟甲喹(FLU)为原料,合成4个碳原子手臂的半抗原(FLUABA),采用活泼酯法与牛血清白蛋白(BSA)偶联制备免疫抗原,通过免疫Balb/c小鼠及细胞融合,获得1株稳定分泌抗氟甲喹单克隆抗体的杂交瘤细胞株DB6-E7,其抗体亚类为IgG1,亲和力常数(KA)为8.19×108L/mol。将氟甲喹、FLUABA及6个碳原子手臂的半抗原FLUACA分别与卵清白蛋白(OVA)偶联作为包被抗原,研究异源包被对间接竞争ELISA灵敏度的影响。结果表明,异源包被可显著提高ELISA方法的灵敏度。基于最佳异源包被(FLU-OVA)的酶联免疫吸附分析法的IC50为26.33μg/L,检出限为4μg/L,定量检测范围为8.0～114μg/L(IC20～IC80)。与喹诺酮类药物及结构类似物几乎不存在交叉反应,特异性高。此方法可满足畜禽产品中氟甲喹残留的快速筛查。
The hapten of Flumequine（ FLU） with four carbon atoms spacer arm（ FLUABA） was synthesized and coupled to bovine serum albumin（ BSA） as immunogen using activated ester method. Balb/c mice were immunized by the artificial immunogen and the splenocytes of immunized mice were fused with Sp2 /0 cells to obtain the monoclonal antibody（ McAbs） . A hybridoma cell line（ DB6-E7） secreting anti-flumequine McAbs was obtained by limited dilution method and screened by indirect enzyme-linked immunosorbent assay （ ELISA） using heterogenous coating antigen. The results showed that the subtype of the McAb was IgG1,and the affinity was 8. 19 × 108 L/mol. The haptens of FLU,FLUABA and FLUACA,with different space arm, were separately linked to ovalbumin（ OVA） for heterologous or homologous coating antigen. The results of indirect ELISA and indirect competitive ELISA （ icELISA） indicated that the heterologous coating antigen could improve the sensitivity of ELISA significantly. By heterologous coating antigen （ FLU-OVA） ,the icELISA showed an IC50 value of 26. 33 μg/L,LOD of 4. 0 μg/L,and the workable range of 8-114 μg/L （ IC20–IC80） . Cross-reactivity studies showed that the McAbs were quiet specific for FLU,no cross-reactivity （ 0. 1%） was detected between the obtained McAbs and the quinolones compounds or other structural similarity compounds. The developed icELISA for FLU can satisfy the detection criteria of flumequine in animal food-products.
出处
《分析化学》
SCIE
EI
CAS
CSCD
北大核心
2010年第3期313-317,共5页
Chinese Journal of Analytical Chemistry
基金
国家自然科学基金(No.30901005)
国家863计划(No.2007AA10Z437)
广东省科技计划项目(No.2009A020101004)
广东省自然科学基金(No.06300421)项目资助
关键词
氟甲喹
单克隆抗体
异源包被
酶联免疫吸附分析法
Flumequine
Monoclonal antibody
Heterogenous antigen
Enzyme-linked immunosorbent assay
