卡那霉素单克隆抗体的研制及ELISA检测方法的建立

摘要

Kanamycin ( KAN) complete antigen, kanamycin-bovine serum albumin ( KAN-BSA) and ka-namycin-chicken ovalbumin ( KAN-OVA) were prepared by EDC. A kanamycin-bovine serum albumin ( KAN-BSA) conjugate was employed as an immunogen to immunize BALB/c mice. A hybridoma cell line stably secreting anti-KAN monoclonal antibody was obtained by hybridoma technique ( 1E1). The monoclonal antibody was prepared by vivo induced ascites method, and the monoclonal antibody was identified. The titer was 8×105. The affinity was 2.8×109 L/mol, and the half-inhibitory concentration ( IC50) was 1.09 ng/mL. The cross-reactivity of Tobmycin ( TOB) was 53.4%, and it was not cross-re-active with other aminoglycoside compounds. Using this monoclonal antibody, a KAN indirect competitive rapid enzyme-linked immunosorbent assay ( ciELISA) was established. The recovery of KAN in milk by ciELISA was 64.4%～89.6%, and the minimum detection limit was 1.92 ng/mL.%采用EDC法制备卡那霉素(KAN)完全抗原、卡那霉素-牛血清白蛋白(KAN-BSA)、卡那霉素-鸡卵清蛋白(KAN-OVA).用KAN-BSA完全抗原免疫BALB/c小鼠,并通过杂交瘤技术制备能够稳定分泌抗KAN单克隆抗体的杂交瘤细胞(1E1).通过体内诱生腹水大量制备单克隆抗体,鉴定1E1单克隆抗体,其效价为8×105,亲和力为2.8×109 L/mol,半数抑制浓度(IC50)为1.09 ng/mL,与妥布霉素(TOB)的交叉反应率为53.4%,与其他氨基糖苷类化合物没有交叉反应性.利用该单克隆抗体,建立KAN间接竞争酶联免疫吸附试验(ciELISA).ciELISA检测KAN在牛奶中的回收率为64.4%～89.6%,最低检测限为1.92 ng/mL.