恩诺沙星抗体免疫检测及其分子识别机制研究

Immunodetection and Molecular Recognition Mechanism of Antibodies against Enrofloxacin

摘要:

兽药恩诺沙星对人体具有较强的毒副作用，检测恩诺沙星残留对保障食品安全具有重要的作用。本研究将恩诺沙星与牛血清白蛋白（BSA）的偶联物注射免疫BALB/c小鼠制备恩诺沙星单克隆抗体，并建立基于单克隆抗体的酶联免疫法检测样品羊奶中恩诺沙星的方法。将筛选出的杂交瘤细胞注射小鼠提取腹水，并以此单克隆抗体建立直接竞争ELISA方法。其半数抑制率（IC50）为0.71±0.05 ng/mL，最低检测限（IC15）为0.04±0.02 ng/mL。检测羊奶样品含恩诺沙星在10~200 ng/mL时回收率为96.56~105.10%，且与HPLC法检测呈良好线性关系（R2=0.9998）。最后利用计算机生物信息学构建恩诺沙星抗体的可变区三维结构并与抗原进行分子对接。结果显示Arg98、Asp99、Gly101、Thr50、Ser51、Ala82、Tyr85等氨基酸残基在抗体抗原识别过程中起关键作用。这些信息对今后抗体结构修饰具有理论指导意义。

Abstract:

Enrofloxacin (ENR) is a veterinary drug with strongly toxic side effects in humans. Therefore, the detection of ENR in food has important implications for food safety. Anti-ENR monoclonal antibody (mAB) was prepared by immunizing BALB/c mice with conjugates of ENR and bovine serum albumin (BSA). The resulting monoclonal antibody was used to establish a direct competitive enzyme-linked immunoabsorbent assay (ELISA) to detect ENR in goat milk samples. The limit of the detection (IC15) was 0.04 ± 0.02 ng/mL and half inhibition rate (IC50) was 0.71 ± 0.05 ng/mL. The recovery rates were 96.56% to 105.10% when ENR concentrations were between 10 and 200 ng/mL in goat milk. ELISA and high-pressure liquid chromatography (HPLC) showed a good linear correlation (R2=0.9998). Finally, the 3-dimentional (3D) structure of the ENR antibody variable region was modelled and docked using computer bioinformatics. The results showed that Arg98, Asp99, Gly101, Thr50, Ser51, Ala82, and Tyr85 performed key functions during antibody-antigen recognition. The results of this study provide theoretical guidance for future antibody structure modifications.