单增李斯特菌单克隆抗体的研制及特性分析

Preparation and Characterization of Monoclonal Antibodies Against Listeria monocytogenes

运用辐照法和热灭菌法自制单增李斯特菌的免疫原、检测原,运用尾静脉免疫方法免疫BALB/c小鼠,运用杂交瘤技术进行细胞融合,制备得到抗单增李斯特菌的单克隆抗体,并对其进行免疫学特性分析。结果表明,成功筛选4株能稳定分泌抗单增李斯特菌的单克隆杂交瘤细胞株,腹水抗体效价为1∶102 400～1∶409 600,免疫球蛋白亚型为Ig G1、Ig2a、Ig2b,亲和力常数在1×107～1×1010L/mol;经测定分析出最佳配对抗体;并与绵羊李斯特菌、英诺克李斯特菌及大肠杆菌、沙门氏菌、枯草杆菌、金黄色葡萄球菌、链球菌等菌属无明显交叉反应;进行双抗体夹心ELISA方法对模拟单增李斯特菌污染肉样检测其灵敏度达1×103 CFU/m L。获得高效价、特异性强、灵敏度高的单克隆抗体,为食品中致病菌单增李斯特菌残留的免疫学检测方法奠定了基础。

In order to explore the specific monoclonal antibodies against Listeria monocytogenes for the immunological detection, irradiation life-style and thermal extinguishing method were used to produce Listeria nonocytogenes antibodies as the antigen,four hybridomas stably secreting Mc Abs to Listeria monocytogenes were developed. The results showed that fourhybridomas were successfully established,and the indirect ELISA titers of the ascites were 1：104～1：4 ×104. Their immune globulin subtypes were Ig G1,Ig2 a,Ig2b.Therefore,Listeria monocytogenes could be detected by a two-antibody-sandwich enzyme-linked immunosorbent assay（das-ELISA） formed by Mc Abs. The affinity constants（Kaffi） were from 1 ×107L/mol to 1 ×1010L/mol,which had no cross-reaction between the other compounds. The sensitivity of LM Mc Abs in the detection of antigen was as low as1×103CFU/m L. The high-titer,sensitive and specific anti-LM monoclonal antibodies were produced in this study,which makes it possible to develop immunoassay for detection of LM residues.