赤点石斑鱼免疫球蛋白单克隆抗体的制备及其特性分析

Production and Characterization of Monoclonal Antibodies Against Epinephelus akaara Ig

赤点石斑鱼血清免疫球蛋白经饱和硫酸铵沉淀,Sephrose-4B柱提纯后免疫BALB/c小鼠。经细胞融合和抗体分泌细胞筛选,获得2株抗石斑鱼免疫球蛋白的单克隆抗体,分别命名为Mab-8D6和Mab-2D3,两株单抗的亚级份均为IgG1,腹水抗体的ELISA效价为106,对纯化的石斑鱼免疫球蛋白的检测灵敏度为62ng,其中Mab-2D3只与石斑鱼免疫球蛋白发生特异性结合,而Mab-8D6除了与石斑鱼免疫球蛋白产生反应外,还能与褐菖鲉、鲈鱼、大黄鱼等海水鱼血清免疫球蛋白发生交叉反应,与锦鲤、欧洲鳗鲡血清免疫球蛋白产生微弱交叉反应。2株单抗Western-blotting反应特性相同,都只能识别非变性非还原条件下790kD大小的石斑鱼免疫球蛋白,而不能识别在变性条件下石斑鱼免疫球蛋白的重链和轻链。以上结果表明,Mab-2D3和Mab-8D6两株单抗所对应的抗原表位不同,Mab-2D3特异性强,所对应的抗原表位是石斑鱼免疫球蛋白所特有的;而Mab-8D6交叉反应结果提示石斑鱼与被测的几种海水鱼的免疫球蛋白进化亲缘关系较近。

Immunoglobulin of red spotted grouper（Epinephelus akaara）,purified by saturation ammonium sulfate precipitation follow with Sephrose-4B chromatography,was used as antigen to immunize BALB/c mice.After cell fusion and screening the antibody secreting cell,two hybridoma cell lines which secreting monoclonal antibodies（Mab） against E.akaara Ig had been established.In isotyping analysis,the results showed that both Mabs were IgG1.ELISA results showed that titer of ascites was 106 and sensitivity of the Mabs to purified E.akaara Ig was as low as 62ng.Further experiments proved that Mab-2D3 only specifically bound to the Ig of E.akaara,while Mab-8D6 not only able to recognize the Ig of E.akaara but also recognizes serum Ig of some marine fishes such as Cuvier et Valenciennes,Lateolabrax japonicus,Pseudosciaena crocea.Mab-8D6 also had low level cross reaction with the serum Ig of Cyprinus carpio and Anguilla anguilla.Western-blotting revealed that both Mabs can recognized molecular weight 790 kD Ig of E.akaara under non-denatured and non-reduced conditions,while neither of the Mabs can recognized the heavy chain and light chain of E.akaara Ig under denatured conditions.All the results demonstrated that Mab-2D3 and Mab-8D6 against different epitopes,Mab-2D3 have stronger specificity and recognize unique epitopes of E.akaara Ig,while cross reaction result of Mab-8D6 suggested that E.akaara have closely evolution relation on Ig with above marine fishes.