环丙沙星单克隆抗体的制备及鉴定

通过改进的碳二亚胺法制备环丙沙星- 牛血清白蛋白偶联物(CIP-BSA)，用紫外扫描、十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)方法鉴定偶联物。利用制备的CIP-BSA 偶联物作为免疫原，免疫Balb/c 小鼠，用杂交瘤技术制备环丙沙星单克隆抗体，并对其效价、亲和力和特异性进行鉴定。结果表明：CIP-BSA 的偶联比为30:1。经间接酶联免疫吸附(ELISA)法筛选，共筛出两株敏感、特异的杂交瘤细胞(JY-1、JY-2)。这两株细胞的腹水经纯化后的抗体效价分别为5.12 × 106、2.56 × 106，亲和力常数分别为2.88 × 109、2.46 × 109L/mol，两抗体均为IgG1a，取亲和力高的JY-1 进行竞争抑制测定，IC50 值可以达到9.83ng/mL。该单抗仅与恩诺沙星存在交叉反应，交叉反应率为95%。通过验证得到的JY-1 细胞株分泌的单克隆抗体可用于水产品中环丙沙星残留的快速检测。

Preparation and Identification of Monoclonal Antibody against Ciprofloxacin

The ciprofloxacin-bovine serum albumin conjugate (CIP-BSA) was prepared by a modified carbodiimide method, and identified by ultraviolet spectrometry and polyacrylamide gel electrophoresis (SDS-PAGE). Then CIP-BSA was used as immunogen to immunize BALB/c mice, inducing the secretion of antibodies against hapten CIP. The monoclonal antibodies (Mabs) against CIP were obtained by hybridoma technology and their titer, affinity and specificity were identified. The results showed that CIP-BSA coupling ratio was 30:1, and two Mabs secreted by cell lines JY-1 or JY-2 belonging to IgG1a were screened by the indirect ELISA method. The titers of purified JY-1 and JY-2 Mabs were 5.12 × 106 and 2.56 × 106, respectively; both the Mabs showed high affinity to CIP with affinity coefficients of 2.88 × 109 and 2.46 × 109 L/mol respectively. A competitive inhibition curve of the JY-1 Mab was developed, and the IC50 was 9.83 ng/mL. The JY-1 Mab exhibited high cross-reactivity (95%) with enrofloxacin. These results suggested that the JY-1 Mab was useful for determination of CIP residues in aqua-products.