间接竞争ELISA检测蜂蜜中氧氟沙星残留

杨兴东; 魏慧雅; 曲扬

doi:10.13386/j.issn1002-0306.2023120006

间接竞争ELISA检测蜂蜜中氧氟沙星残留

Determination of Ofloxacin Residues in Honey by Indirect Competition ELISA

摘要

摘要: 为了建立间接竞争ELISA法检测氧氟沙星（Ofloxacin，OFLX）在蜂蜜中的残留，本研究采用活性酯法将OFLX与载体蛋白偶联，得到抗OFLX完全抗原（OFLX-BSA）和检测抗原（OFLX-OVA）。采用OFLX-BSA免疫BALB/c小鼠，之后用杂交瘤等技术制备抗OFLX单克隆抗体。通过优化反应条件，建立间接竞争ELISA方法，并对该法的准确度、精密度和特异性进行判定。结果显示OFLX与载体蛋白偶联成功；得到一株抗OFLX的杂交瘤细胞株（3D7），单克隆抗体的IC₅₀值为1.17 ng/mL；该法在蜂蜜中的OFLX平均添加回收率为84.1%，其批间变异系数均大于批内的变异系数，与马波沙星的交叉反应率为52.9%，与其他竞争反应物没有交叉反应。本研究建立的间接竞争ELISA方法能够满足OFLX在蜂蜜中残留的测定需求。

Abstract: In order to establish an indirect competitive ELISA for the detection of ofloxacin (OFLX) residues in honey, in this study, OFLX was coupled to carrier proteins using the active ester method to obtain anti-OFLX complete antigen (OFLX-BSA) and detection antigen (OFLX-OVA). BALB/c mice were immunized with OFLX-BSA, after which anti-OFLX monoclonal antibodies were prepared by hybridoma and other techniques. An indirect competitive ELISA method was developed by optimizing the reaction conditions and the accuracy, precision and specificity of the method were determined. The results showed that OFLX was successfully coupled to the carrier protein; a hybridoma cell line (3D7) against OFLX was obtained. And the IC₅₀ value of the monoclonal antibody was 1.17 ng/mL. The average recovery rate of OFLX spiked in honey by this method was 84.1%, and its intra-batch coefficients of variation were greater than inter-batch ones in all batches. The cross-reaction rate with marbofloxacin was 52.9%, and no cross-reaction with the other competing reactants. The indirect competitive ELISA method developed in this study is able to fulfill the requirements for determination of OFLX residues in honey.