七种常见宠物二级抗体的制备和HRP标记

Preparation and HRP Labeling of Rabbit Polyclonal Antibodies against Seven Pet Animals

目的制备兔抗7种中国家庭中常见宠物的二级抗体,并进行辣根过氧化酶标记,为宠物血清学检测系统的建立提供工具。方法利用饱和硫酸铵沉淀法粗纯抗体后,再利用protein A 或protein G亲和层析的方法进一步纯化宠物的IgG,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宠物的二级抗体,采用简易过碘酸钠法对纯化的兔抗宠物的二级抗体进行HRP标记,通过ELISA方法测定标记抗体的效价,并利用Western blotting考察标记抗体的特异性。结果纯化了狗、家猫、豚鼠、金仓鼠、松鼠、花鼠和龙猫7种宠物的血清IgG,免疫双扩散法测定兔抗这7种宠物的抗血清效价均达到1:32,并对纯化的兔抗7种宠物的二级抗体进行了HRP标记,ELISA测定标记抗体的效价达到1:(2000～15000)左右,Western blots显示标记抗体具有很好的特异性。结论成功制备了HRP标记的兔抗7种中国家庭中常见宠物的二级抗体,为宠物血清学检测体系的建立提供了工具。

Objective To prepare the rabbit polyclonal antibodies against seven pet animals, which were commonly kept in Chinese family, and to label the secondary antibodies with horseradish peroxidase for developing the serological test system of pet animals. Methods IgGs of pet animals were purified by precipitation with ammonium sulfate combining affinity chromatography using Protein A and Protein G. Rabbit anti-sera were prepared by repeatedly immunized rabbits with purified IgGs. The antibodies against pet animal IgG in rabbit sera were detected by agarose double immunodiffusion and purified using Protein A affinity chromatography. The products were labeled using HRP by periodate oxidation method. And the activities of the labeled rabbit polyclonal antibodies against seven pet animals were detected by ELISA. Western blots were performed to investigate the specificity of HRP-IgG against the IgG of pet animals. Results The IgGs from the seven pet animals species were purified, including dog, cat,guinea pig, golden hamster, squirrel, western chipmunk, chinchilla. The titers of seven rabbit anti-sera obtained up to 1：32 examined by agarose double immunodiffusion. Seven kinds of purified rabbit anti-pet secondary antibodies were labeled with HRP and their activities were up to 1：（2000 - 15000） examined by ELISA. The specific activities of the seven rabbit anti-pet IgGsHRP were indicated with western blot. Conclusion Rabbit polyclonal antibodies against seven pet animals labeled with HRP were prepared successfully, it was an useful tool for developing the serological test system of pet animals, which were commonly kept in Chinese family.