九种重要经济动物二级抗体的制备和HRP标记

目的制备兔抗9种重要经济动物的二级抗体,并进行辣根过氧化酶标记,为经济动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动物血清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亲和层析的方法纯化兔抗经济动物的二级抗体,采用简易过碘酸钠法对纯化的二级抗体进行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。结果纯化了家猪,绵羊、山羊、牛、马、驴、狐狸、貉和黑貂9种重要经济动物的血清IgG,免疫双扩散法测定兔抗血清效价均达到1：32,并对纯化的兔抗家猪,绵羊、山羊、牛、马、驴、狐狸、貉和黑貂9种重要经济动物二级抗体进行了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9种重要经济动物的二级抗体,为经济动物血清学检测体系的建立提供了工具。

Preparation and HRP Labeling of Rabbit Polyclonal Antibodies against Nine Important Economical Animals

Objective To prepare the rabbit polyclonal antibodies against nine economical animals, and to label the secondary antibodies with horseradish peroxidase for developing the serological test system for economical animals. Methods IgGs of economical 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 economical animals 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 antibodies were detected by ELISA. Western blots were performed to investigate the specificity of HRP-IgG against the IgG of economical animals. Results IgGs of nine economical animals species were purified, including pig, sheep, goat, cattle, horse, donkey, fox, raccoon dog, sable. The titers of nine rabbit anti-sera obtained up to 1：32 examined by agarose double inununodiffusion. Nine kinds of purified rabbit anti-economical animals IgGs were labeled with HRP and their activities were up to 1 ： （2000 - 15000） examined by EL/SA. The specific activities of the nine rabbit anti-economical animals IgGs-HRP were indicated with westem blots. Conclusions Rabbit polyclonal antibodies against nine economical animals labeled with HRP were prepared successfully, and this provide a useful tool for developing the serological test system of economical animals.