鸡白细胞介素-6原核表达及双抗夹心ELISA方法的建立

Prokaryotic expression of chicken interleukin-6 gene and establishment of double sandwich ELISA

应用分子生物学技术原核表达ChIL-6,以ChIL-6重组蛋白为免疫原,按免疫程序分别制备兔抗和鼠抗IL-6重组蛋白的多克隆抗体。应用此抗体建立双抗夹心ELISA方法后,为了优化此方法本试验对该方法的最佳试验条件、标准曲线、重复性和初步应用进行确定。结果显示,经SDS-PAGE和Western-blot分析,表明ChIL-6在大肠杆菌中正确表达,为了建立检测ChIL-6的双抗夹心ELISA方法,本试验应用表达的重组蛋白制备兔抗和鼠抗多克隆抗体。建立的双抗夹心ELISA方法最佳反应条件为包被抗体的质量浓度为50mg/L,4℃过夜;酶标二抗稀释度为1∶400,37℃1h;应用该方法检测感染金黄色葡萄球菌后的ChIL-6,其结果与本实验室之前应用人的ELISA试剂盒检测的结果相似。结果表明,本试验建立的检测ChIL-6的双抗夹心ELISA方法可用于临床。

The study appled ChlL-6 recombinant protein to prepare polyclonal antibodies of rabbit anti-ChlL-6 and mouse anti-ChlL-6, established double-antibody sandwich ELISA method though application of two antibodies, deter- mined the best experimental conditions, standard curve, repeatability and preliminary applications of the method. SDS-PAGE and Western-blot of the recombinant protein showed that ChlL-6 gene was expressed in E＇. coli success- fully. The recombinated protein was applied to immunize rabbit and BALB/c for serum production of anti-ChlL 6. The optimal reaction conditions of the double-antibody sandwich ELISA was 50 mg/L of coating antibody concentra- tion,4~C overnight;and enzyme linked second antibody dilution was 1 ： 400,37~C 1 h. Using the method to test ChlL-6 which infected with Staphylococcus aureus ,its results was similar to that of other ELISA test kits detecting ChlL-6. The double-antibody sandwich ELISA for detection of ChlL-6 which established in this study can be used in clinic.