氟虫腈单克隆抗体的制备及其在牛乳检测中的应用

Preparation of Monoclonal Antibody against Fipronil and Its Application for Fipronil Detection in Milk

合成氟虫腈人工完全抗原,制备氟虫腈单克隆抗体,以氟虫腈及氟虫腈类似物为半抗原,分别采用戊二醛法和碳二亚胺法合成人工完全抗原;利用紫外光谱扫描法、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法进行鉴定;使用氟虫腈-牛血清蛋白人工完全抗原免疫BALB/c小鼠,得到杂交瘤细胞株,采用小鼠体内诱生腹水方法制备单克隆抗体;使用酶联免疫吸附测定法测定氟虫腈在牛乳中的加标回收率及精密度。结果表明:氟虫腈的人工完全抗原合成成功,制备的单克隆抗体3F6质量浓度为8.5 mg/mL,效价为2.0×10^(6),亚型为IgG1,与氟甲腈、氟虫腈砜、氟虫腈亚砜的交叉反应率分别为9.21%、14.02%、21.46%;氟虫腈在牛乳中的加标回收率为87%~118%,变异系数低于15%,方法具有较高准确度和精密度。

In this study,the complete antigens of fipronil and its analogue were synthesized using glutaraldehyde(GA)and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC)as a cross-linker,respectively,and were identified by ultraviolet absorption spectroscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).BALB/c mice were immunized with fipronil-bovine serum albumen to obtain hybridoma cell 3F6.Monoclonal antibodies were prepared by inducing ascites in mice.An enzyme-linked immunosorbent assay(ELISA)was developed and evaluated to determine fipronil in milk.The results showed that the concentration of the prepared monoclonal antibody,belonging to the IgG1 subclass,was 8.5 mg/mL,and the titer was 2.0×10^(6).The cross-reaction rates with fipronil-desulfinyl,fipronil sulfone and fipronil sulfoxide were 9.21%,14.02%and 21.46%,respectively.The recoveries of fipronil in spiked milk were 87%–118%,and the coefficient of variation for precision was less than 15%.In conclusion,this method is highly accurate and precise.