测定小鼠细胞因子的双抗体夹心ELISA法的建立及应用研究

目的　建立敏感的测定小鼠IL - 5、IL - 4和IFN -γ水平的生物素 -链霉亲和素双抗体夹心ELISA法 ,并探讨该法在IL - 5转基因小鼠感染模型中的应用价值。方法　含大鼠抗小鼠IL - 4、IL - 5和IFN -γ单克隆抗体的杂交瘤细胞培养上清 ,经PM 10膜超滤、5 0 %饱和硫酸铵沉淀及ProteinG -Sepharose亲和层析纯化。应用活化生物素制剂对纯化的TR FK4、BVD6和AN18单克隆体抗体进行生物素化。用纯化的 2 μg/mlTRFK5、8μg/ml 11B11和 4 μg/mlR4 - 6A2分别作为测定小鼠IL - 5、IL - 4和IFN -γ的包被抗体 ,然后逐步加入样品、生物素化TRFK4 (1μg/ml)、BVD6 (2 μg/ml)和AN18(1μg/ml)、SA -HRPO和OPD ,硫酸终止反应后读取A490 值。结果　所建立的生物素 -链霉亲和素双抗体夹心ELISA法特异性和敏感性高 ,用于检测感染巴西日圆线虫感染的IL - 5转基因小鼠和非转基因小鼠的血清标本取得了较好的效果。结论　该ELISA方法对于测定小鼠细胞因子具有应用价值

Establishment of double antibody sandwich ELISA for mouse cytokines and its application

Aim To establish a sensitive biotin strepavidin double antibody sandwich ELISA for mouse IL 5,IL 4,IFN γ and to explore its application in IL-5 transgenic mice infected with Nippostrongylus brasiliensis.Method The culture supernatants of hybridoma were purified by ultrafiltering with PM10 membrane,precipitating with 50% of saturated ammonium sulfate and Protein G Sepharose affinity column.Purified TRFK4,BVD6 and AN18 McAbs were biotinylated with the biotinylation reagent.2μg/ml of TRFK5,8μg/ml of 11B11 and 4μg/ml of R4-6A2 were as coating antibodies of detecting IL-5,IL-4 and IFN-γ,separately,Then the samples,biotinylated TRFK4(1μg/ml),BVD6(2μg/ml) and AN18(1μg/ml),SA HRPO and OPD were added.Finally,the reaction was stopped by sulfuric acid and A 490 value was read. Results The method had a high specificity and sensitivity.The promising results have been obtained for detecting the levels of cytokines of sera from IL 5 transgenic mice and non transgenic mice infected with N.brasiliensis.Conclusion The method can be used to detect mouse cytokines.