ELISA方法研究多糖与人干扰素-γ相互作用

Interaction between polysaccharides and interferon-gamma using an improved ELISA approach

目的 :建立一种体外研究多糖与干扰素 - γ(Interferon- γ,IFN- γ)相互作用的 EL ISA方法。方法 :在硼氰氢化钠作用下合成肝素 -牛血清白蛋白复合物 (Heparin- BSA Complex,HBC) ,Sepharose4 B凝胶过滤系统分离纯化 HBC,并用 SDS- PAGE鉴定。将 HBC(相当于 0 .5 μg蛋白含量 )包被在酶标板上 ,EL ISA方法检测 HBC与 IFN- γ的相互作用 ,并探讨游离肝素、低分子量肝素、硫酸软骨素 A、硫酸软骨素 C和透明质酸 ,以及卡拉胶等多糖对这种结合的影响。结果 :IFN- γ与 HBC的结合呈 IFN- γ剂量依赖性 ,2 ng左右达到饱和。游离肝素和低分子量肝素以及其他多糖均不同程度地抑制了 IFN- γ与 HBC的结合 ,肝素和低分子肝素的 IC50 分别为 2 .4 μg/ ml和 18.6 μg/ml。结论 :IFN- γ与肝素、卡拉胶高亲和力结合 ,而与硫酸软骨素 A、硫酸软骨素 C、透明质酸结合力极弱。EL ISA是一个简单。

Objective: To establish an ELISA approach to study the interaction of polysaccharides with cytokine in vitro. Methods: The heparin-BSA complexes(HBC)were synthesized with a chemistry method and separated (using) a 1×90 cm column of Separose 4B. After identification of the complex via SDS-PAGE,the wells of ELISA plates were coated with HBC and the interaction of HBC with interferon-gamma (IFN-γ) was detected. The effects of heparin,low molecular weight heparin (LMW heparin),chondroitin sulfate(CS),hyaluronic acid(HA)and carrageenans on the binding of HBC to IFN-γ were tested in this system. Results: Human recombinant IFN-γ bound to heparin in a concentration-dependent manner,the binding of IFN-γ to HBC was detected at the concentration of 0.25 ng,and saturated at around 2 ng. Free heparin,LMW heparin,CS,HA and carrageenans competed for the binding of IFN-γ to HBC with significant different ability. The IC_(50) concentrations of heparin and LMW heparin were 2.40 μg/ml and 18.60 μg/ml respectively. Conclusion: IFN-γis a cytokine with high binding affinity to (heparin) and carrageenans family but poor to CS-A and CS-C. ELISA is a simple,sensitive approach to detect the (interaction) of polysaccharides with cytokine in vitro.