GLP-1衍生物多克隆抗体的制备及应用

Preparation and application of polyclonal antibody against GLP-1 derivative

[目的]制备抗LmGLP-1的高特异性多克隆抗体,为LmGLP-1的活性和应用研究提供基础。[方法]以高纯度LmGLP-1为免疫原,对SD大鼠进行皮下注射免疫,获得抗血清,通过Western Blotting鉴定其特异性,采用ELISA测定多抗效价,建立间接ELISA测定LmGLP-1的工作曲线,并验证多抗在免疫沉淀(IP)和间接ELISA测定大鼠血样中LmGLP-1抗原的应用。[结果]获得的抗血清能特异性识别利用原核表达系统制备的LmGLP-1抗原,效价高达1∶16 000,建立的间接非竞争ELISA工作曲线在0.50~80 ng/mL范围内线性良好,检出限为0.25 ng/mL,能有效测定复杂血样中LmGLP-1的浓度。免疫沉淀反应显示,抗血清能识别大鼠体内的外源性LmGLP-1,也可识别低水平的内源性GLP-1。[结论]所制备的LmGLP-1抗血清具有良好的反应性和特异性,为后续LmGLP-1的机制研究和药效、药代动力学的开展提供了基础。

[Objective]To prepare highly specific polyclonal antibody against LmGLP-1 for the activity and application researches.[Method]The SD rats were immunized with high purity LmGLP-1 subcutaneously to obtain antiserum. The specificity of the antiserum was identified by Western Blotting. The titer of the polyclonal antibody was determined by ELISA. The working curve of indirect ELISA was established. The application of polyclonal antibodies in the determination of LmGLP-1 in rat blood samples by immunoprecipitation(IP) and indirect ELISA was tested. [Result]The obtained polyclonal antibody could specifically recognize LmGLP-1 in blood samples,and the titer was as high as 1∶16 000. The established indirect ELISA working curve showed good linearity in the range of 0. 5-80 ng/mL with a detection limit of 0. 25 ng/mL. Immunoprecipitation reaction showed that the antiserum could recognize endogenous GLP-1 as well as exogenous LmGLP-1 in rats. [Conclusion]The LmGLP-1 antiserum prepared in this study has good reactivity and specificity,which provides the foundation for subsequent mechanism,pharmacodynamics and pharmacokinetics researches of LmGLP-1.