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为自主研制抗恩诺沙星的高亲和力、高特异性单克隆抗体,提供动物源食品中恩诺沙星药物残留生物学快速检验方法试剂,利用碳二亚胺法(carbodiimide,EDC)制备恩诺沙星(enrofloxacin,ENR)的人工抗原,免疫Balb/c小鼠,采用PEG融合法,通过有限稀释法筛选出抗恩诺沙星的单克隆抗体,获得了两株抗恩诺沙星的单克隆抗体(克隆号:6A4和8D6)。经过离子交换法纯化得到的抗体纯度在95%以上,其中,纯化后的6A4抗体在1mg/mL时,效价达到1.28×10-5,8E6抗体在1mg/mL时,效价达到1.024×10-6。两株单克隆抗体与环丙沙星的交叉反应率为1.5%,与其他氟喹诺酮类药物不反应。在酶联免疫法(enzyme-linked immunosorbent assay,ELISA)中,6A4和8E6抗体的检测限分别为3.125μg/kg和0.3μg/kg。采用的半抗原合成以及单克隆抗体筛选的方法可以获得高灵敏度、高特异性的抗恩诺沙星的单克隆抗体,经后续研究证实单克隆抗体(8E6)可以用于免疫胶体金和酶联免疫法检测食品中残留的恩诺沙星。
In order to independently develop high affinity and high specificity monoclonal antibodies to enrofloxacin, we provide reagents for the rapid biological assay of enrofloxacin residues in animal foodstuffs, and carbodiimide (EDC) Balb / c mice were immunized with the artificial antigen of enrofloxacin (ENR). Monoclonal antibody against enrofloxacin was screened by limiting dilution method by PEG fusion method. Two anti-enrofloxacin Monoclonal antibodies (clone number: 6A4 and 8D6). The purity of the purified antibody was above 95% by ion exchange method. The titer of the purified 6A4 antibody reached 1.28 × 10-5 at 1mg / mL and the titer reached 1.024 × 10 at 1mg / mL -6. The cross-reactivity of the two monoclonal antibodies with ciprofloxacin was 1.5%, not reacting with other fluoroquinolones. The detection limits of the 6A4 and 8E6 antibodies in the enzyme-linked immunosorbent assay (ELISA) were 3.125μg / kg and 0.3μg / kg, respectively. Monoclonal antibodies against enrofloxacin with high sensitivity and specificity can be obtained by using hapten synthesis and monoclonal antibody screening. After further studies, monoclonal antibody (8E6) can be used to immunogold and enzyme conjugate Immune detection of residual enrofloxacin in food.