沙鼠IgG兔抗血清的制备及其在IgG抗体检测中的应用

Preparation and application of rabbit antiserum against purified Mongolian gerbil IgG

目的用纯化的蒙古沙鼠血清IgG制备相应兔抗血清,并用于幽门螺杆菌(Helicobacter pylori,Hp)疫苗免疫后沙鼠血清中抗原特异性IgG的检测。方法采用饱和硫酸铵沉淀法和Q sepharose high performance阴离子交换层析法纯化蒙古沙鼠血清IgG,免疫日本大白耳兔制备兔抗沙鼠IgG抗血清并进行rProtein A亲和纯化,建立间接ELISA方法用于检测Hp疫苗注射免疫蒙古沙鼠后血清抗原特异性IgG水平。结果以纯化的蒙古沙鼠血清IgG(纯度大于98%)免疫日本大白耳兔获得相应兔抗血清,经亲和层析后其纯度大于90%,回收率约为85%,双扩效价为1:16,ELISA效价为1:320 000;ELISA检测结果显示Hp疫苗注射免疫蒙古沙鼠于第2次免疫后产生高水平IgG,第4次免疫后第7天IgG水平达到高峰,随后几周仍然保持高水平。结论所制备的兔抗沙鼠IgG抗体可以用于蒙古沙鼠血清中抗原特异性IgG的检测。

Objective To prepare rabbit antiserum against purified Mongolian gerbil IgG and to detect antigen-specific IgG level in the serum of Mongolian gerbil administrated with Helicobacter pylori （Hp） vaccine. Methods Mongolian gerbil serum IgG was purified by ammonium sulfate precipitation and Q sepharose high performance anion exchange chromatography. Rabbit antiserum was prepared by immunizing rabbit with pure Mongolian gerbil IgG, and then purified by rProtein A affinity chromatography. The antigen-specific IgG level was detected with the rabbit antiserum by indirect ELISA. Results Rabbit antiserum was obtained after rabbit was administrated with purified Mongolian gerbil IgG （purity 〉 98%）. Rabbit anti-Mongolian gerbil IgG has high titer level （1 ： 16 in double immunodiffusion test or 1 ： 320 000 in ELISA）, high purity （more than 90%）, and high recovery rate （approximately 85%）. The ELISA result showed that the high IgG level was induced after Mongolian gerbil was immunized with Hp vaccine and the IgG level reached its peak 7 at days after four immunizations. Conclusion The rabbit antiserum to Mongolian gerbil IgG can be used in ELISA to detect antigen-specific IgG level.