兔抗五种鱼免疫球蛋白抗体的制备和HRP标记

目的制备兔抗鱼类免疫球蛋白抗体并进行辣根过氧化酶标记,为鱼类血清学检测系统的建立提供工具。方法利用proteinA亲和层析的方法纯化鱼血清免疫球蛋白,免疫大耳白兔制备抗血清,利用免疫双扩散来检测抗血清的效价,并用Protein A亲和层析的方法来纯化兔抗鱼免疫球蛋白的抗体,采用简易过碘酸钠法对纯化的兔抗鱼免疫球蛋白的抗体进行HRP标记,通过ELISA方法测定标记抗体的效价,并利用Western blotting来考察标记抗体与其他常见鱼类血清蛋白间的交叉反应。结果纯化了鳙、鲤、乌鳢、黄鳝、鲈五种鱼血清免疫球蛋白,免疫双扩散法测定兔抗这五种鱼免疫球蛋白的抗血清效价均达到1∶32,并对纯化的兔抗鳙、鲤、乌鳢、黄鳝、鲈五种鱼类免疫球蛋白的抗体进行了HRP标记,ELISA测定标记抗体的效价达到1∶10000左右,Western blots显示标记抗体与部分其他鱼类免疫球蛋白之间存在不同程度的交叉反应。结论成功制备了HRP标记的兔抗鱼类免疫球蛋白抗体,为鱼类血清学检测体系的建立提供了工具。

Production and HRP Labeling of Rabbit Anti-fish Immunoglobulins Antibodies

Objective To prepare the rabbit anti-fish immunoglobulins antibodies labeled with horseradish peroxidase,and to provide a tool for developing the serological test system of fish. Methods Immunoglobulins of fish were purified by affinity chromatography using staphylococcal protein A. Rabbit anti-sera were prepared by repeatedly immunized rabbits with purified immunoglobulins. And anti-fish immunoglobulins antibodies in rabbit sera were detected by agarose double immunodiffusion. Five rabbit anti-fish immunoglobulins antibodies were purified using staphylococcal protein A affinity chromatography. The products were labeled using HRP by pefiodate oxidation method. And the activities of the labeled rabbit anti-fish immunoglobulins antibodies were detected by ELISA. Western blots were performed to investigate the cross-reactivity of various labeled antibodies with serum proteins from other fish species. Results Immunoglobulins of five fish species were purified, including bighead carp, carp, argus snakehead fish, ricefield eel, sea bass. The titers of five rabbit anti-sera obtained up to 1：32 examined by agarose double immunodiffusion. Five kinds of purified rabbit anti-fish immunoglobulins antibodies were labeled with HRP and their activities were up to 1 ： 10000 examined by ELISA. Four kinds of rabbit anti-fish immunoglobulins antibodies labeled with HRP could react with serum proteins from other fish species. Conclusions Rabbit anti-fish immunoglobulins antibodies labeled with HRP were prepared successfully, and this provide a tool for developing the serological test system of fish.