十七种哺乳动物二级抗体的制备和辣根过氧化物酶标记

Preparation and HRP Labeling of Rabbit Polyclonal Antibodies against Seventeen Mammals

目的制备兔抗17种哺乳动物的二级抗体,并进行辣根过氧化物酶标记,为哺乳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哺乳动物的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哺乳动物的二级抗体,采用简易过碘酸钠法对纯化的兔抗哺乳动物的二级抗体进行辣根过氧化物酶标记,通过ELISA方法测定标记抗体的效价,并利用Western blot方法考察标记抗体的特异性。结果纯化了恒河猴、东北虎、布氏田鼠、黑线姬鼠、斑羚、原驼、果子狸、食蟹猴、梅花鹿、长爪沙鼠、马鹿、骆驼、大仓鼠、豚鹿、熊猴、大耳羊和雪貂等17种哺乳动物的血清IgG,分别免疫大耳白兔制备了这17种哺乳动物的兔抗血清,免疫双扩散法测定兔抗这17种哺乳动物的抗血清效价均达到1∶32,并对纯化的兔抗恒河猴IgG、兔抗东北虎IgG、兔抗布氏田鼠IgG、兔抗黑线姬鼠IgG、兔抗斑羚IgG、兔抗原驼IgG、兔抗果子狸IgG、兔抗食蟹猴IgG、兔抗梅花鹿IgG、兔抗长爪沙鼠IgG、兔抗马鹿IgG、兔抗骆驼IgG、兔抗大仓鼠IgG、兔抗豚鹿IgG、兔抗熊猴IgG、兔抗大耳羊IgG和兔抗雪貂IgG等17种哺乳动物的二级抗体进行了辣根过氧化物酶标记,ELISA测定标记抗体的效价达到1∶(2000～60000)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17种哺乳动物的二级抗体,为哺乳动物血清学检测体系的建立提供了工具。

Objective To prepare the rabbit polyclonal antibodies against seventeen mammals and to label the secondary antibodies with horseradish peroxidase for developing the serological test system of mammals.Methods IgGs of mammals were purified by precipitation with ammonium sulfate combining affinity chromatography using Protein A or Protein G.Rabbit anti-sera were prepared by repeatedly immunized rabbits with purified IgGs.The antibodies against mammal 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 seventeen mammals were detected by ELISA.Western blots were performed to investigate the specificity of the seventeen rabbit anti-mammal IgGs-HRP.Results The IgGs from the seventeen mammal species were purified,including rhesus,tiger,brandt＇s vole,striped hamster,goral,guanaco,civet cat,crab-eating macaque,sika deer,mongolian gerbil,red deer,camel,hog deer,ibex,hamster,assam macaque,ferret.The titers of seventeen rabbit anti-sera were up to 1∶32 examined by agarose double immunodiffusion.Seventeen kinds of purified rabbit anti-mammal secondary antibodies were labeled with HRP and their activities were up to 1∶2000-60000 examined by ELISA.The specific activities of the seventeen rabbit anti-mammal IgGs-HRP were indicated with western blot.Conclusion Rabbit polyclonal antibodies against seventeen mammals labeled with HRP were prepared successfully,it is an useful tool for developing the serological test system of mammals.