十六种鸟类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 Sixteen Birds

目的制备兔抗16种鸟类的二级抗体,并进行辣根过氧化物酶标记,为鸟类血清学检测系统的建立提供工具。方法采用水稀释法粗纯抗体后,再利用改良的饱和硫酸铵分级沉淀法,或亲和层析结合饱和硫酸铵沉淀法,或饱和硫酸铵沉淀法结合SDS-PAGE凝胶切胶纯化的方法进一步纯化鸟类的IgY,利用纯化的IgY免疫大耳白兔制备抗血清,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鸟类的二级抗体,采用简易过碘酸钠法对纯化的兔抗鸟类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 blots考察标记抗体的特异性。结果纯化了灰雁、鸬鹚、鸵鸟、小鹈、鸽子、鹅、孔雀、鹌鹑、贵妃鸡、草鹭、夜鹭、赤嘴潜鸭、燕鸥、长脚鹬、虎皮鹦鹉、翘鼻麻鸭等16种鸟类的IgY,免疫双扩散法测定兔抗这16种鸟类的抗血清效价均达到1∶32,并对纯化的兔抗灰雁IgY、兔抗鸬鹚IgY、兔抗鸵鸟IgY、兔抗小鹈IgY、兔抗鸽子IgY、兔抗鹅IgY、兔抗孔雀IgY、兔抗鹌鹑IgY、兔抗贵妃鸡IgY、兔抗草鹭IgY、兔抗夜鹭IgY、兔抗赤嘴潜鸭IgY、兔抗燕鸥IgY、兔抗长脚鹬IgY、兔抗虎皮鹦鹉IgY、兔抗翘鼻麻鸭IgY等16种兔抗鸟类IgY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∶800～80000左右,Western blots显示标记抗体具有很好的特异性。结论成功制备了辣根过氧化物酶标记的兔抗16种鸟类的二级抗体,为鸟类血清学检测体系的建立提供了工具。

Objective To prepare the rabbit polyclonal antibodies against sixteen stains of birds,and to label the secondary antibodies with horseradish peroxidase for developing the serological test system of birds.Methods IgYs of birds were purified by water diluted,combining precipitation with ammonium sulfate,or affinity chromatography and precipitation with ammonium sulfate,or precipitation with ammonium sulfate and cutting the SDS-PAGE gel slices that contain the IgY bands.Rabbit anti-sera were prepared by repeatedly immunized rabbits with purified IgYs.The antibodies against bird IgY 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 sixteen strains of birds were detected by ELISA.Western blots were performed to investigate the specificity of IgG-HRP against the IgY of birds.Results The IgYs from the strains of birds were purified,including greylag goose,great cormorant,ostrich,little grebe,pigeon,rich bird,goose,peacock,quail,ladyhood-chicken,heron,night-heron,red-crested pochard,tern,black-winged stilt,parrots,common shelduck.The titers of sixteen rabbit anti-sera were up to 1∶32 examined by agarose double immunodiffusion.Sixteen kinds of purified rabbit anti-bird secondary antibodies were labeled with HRP and their activities were up to 1∶800-80000 examined by ELISA.The specific activities of the sixteen rabbit anti-bird IgGs-HRP were indicated with western blot.Conclusions Rabbit polyclonal antibodies against sixteen birds labeled with HRP were prepared successfully;It was an useful tool for developing the serological test system for birds.