Colgalt2-酶联免疫试剂盒的制备

摘要

目的 利用N末端Colgalt2多克隆抗体和C末端Colgalt2多克隆抗体,制备Colgalt2双抗体夹心酶联免疫试剂盒.方法 体外扩增Colgalt2的N-末端编码序列,构建其原核表达质粒,诱导纯化Colgalt2重组蛋白.免疫大耳白兔获得抗Colgalt2 N-末端重组蛋白的多克隆抗体.以Colgalt2 N-末端重组蛋白为抗原,利用ELISA和Western Blot技术检测多克隆抗体效价和特异性.以Colgalt2 N-末端多克隆抗体包被酶标板,同时利用HRP标记的C末端Colgalt2抗体,制备Colgalt2-ElISA试剂盒.结果 我们成功克隆表达了N末端Colgalt2基因片段,制备了兔抗Colgalt2多克隆抗体,ELISA分析表明该抗体效价较高(＞1∶1 280000),Western Blot分析显示该抗体有良好的特异性.对我们所制备的不同浓度的Colgalt2重组蛋白进行分析显示,所制备的分析试剂盒,在1.25～25 μg/ml范围内具有较好分析线性.线性相关系数约为0.98.结论 所制备的Colgalt2-ELISA分析试剂盒可以用于标本内Colgalt2蛋白检测.%Objective To prepare an Colgalt2 ELISA kit using Colgalt2 N-terminal and C-terminal polyclonal antibody.Methods Colgalt2 partial CDs were amplified from HepG2 cell,and inserted into prokaryotic expression plasmids,and induced the expression of N-terminal recombinant Colgalt2 protein.N-terminal Colgalt2 polyclonal antibody was prepared by immunizing rabbits with purified recombinant protein.Titer and specificity of the antibody was determined by ELISA and Western Blot.Microtiter plate was coated with N-terminal Colgalt2 polyclonal antibody,and other polyclonal antibody of Colgalt2 C-terminal was labeled by HRP.Results We successfully cloned N-terminal of Colgalt2 and obtained the recombinant Colgalt2 protein.ELISA analysis showed that titer of anti-Colgalt2 was higher than 1 ∶ 1 280 000.Western Blot analysis showed that the antibody with a good specificity.We successfully developed an ELISA kit of Colgalt2.The data of the different concentrations of recombinant protein Colgalt2 showed that the ELISA kit had a good sensitivity.From1.25 μg/ml to 25μg/ml,the coefficient of linear correlation was 0.98.Conclusion The Colgalt2-ELISA kit may be used to determine Colgalt2 levels in samples.