检测HSA-GLP-1融合蛋白双抗体夹心ELISA方法的建立  

Development of Double Antibodies Sandwich ELISA Method for Quantitative Detection of Fusion Protein HSA-GLP-1

作　　者：王秋实[1] 刘运龙[2] 张玉民[3] 陈知航[2] 钱爱东[1] 程远国[2] 

机构地区：[1]吉林农业大学,吉林长春130118 [2]军事医学科学院微生物流行病研究所,北京100071 [3]延边大学,吉林延吉133000

出　　处：《生物技术通讯》2013年第5期685-690,共6页Letters in Biotechnology

摘　　要：目的：为检测血清中HSA-GLP-1融合蛋白整体分子的浓度，建立一种特异、灵敏的定量检测食蟹猴体内HSA-GLP-1融合蛋白浓度的双抗体夹心ELISA的方法。方法：采用双抗体夹心ELISA方法，以GLP-1单克隆抗体为包被抗体、HSA-GLP-1融合蛋白为夹心抗原、anti-HSA-Biotin为检测抗体，用Streptavidin-HRP进行免疫放大，TMB显色。结果：建立了检测HSA-GLP-1融合蛋白的ELISA方法，其线性范围为15.6~1000 ng/mL，最低检测限为15.6 ng/mL，与GLP-1、HSA、IL2-HSA均无交叉反应，板内和板间精密度均小于15%，准确度为±15%，冻融稳定性和稀释稳定性良好。结论：建立的HSA-GLP-1蛋白检测方法符合新生物制品临床前药代动力学研究指导原则要求，可用于HSA-GLP-1融合蛋白在临床前药代动力学试验的定量检测。Objective： There is no commercial kit for testing HSA-GLP-1 fusion protein, we need to develop a high specific, high sensitive double antibody sandwich ELISA method for the quantification of fusion protein HSA-GLP-1 by method of antibody pairing. Methods： An quantitative sandwich enzyme immunoassay was devel-oped in using goat anti-GLP-1 monoclonal antibody for capturing, and a biotin-labeled of another anti-HSA mono-clonal antibody as well as detecting, and the HRP labeled conjugate streptavidin was used. Following that, color was developed by the TMB solution and the reaction was stopped by stop solution. Results： An ELISA assay was developed with a wide dynamic range of concentrations from 15.6~1000 ng/mL. The lowest quantification of this as-say was 15.6 ng/mL. The specificity assay indicated that it had no cross-reaction with GLP-1, HSA, IL-2/HSA. Both accuracy of the intra- and inter-assay were less than 15%. Conclusion： The assay is highly sensitive, accu-rate, specific, and reproducibility over a wide dynamic range of concentrations, which was proven to be a feasible quantitative method for fusion protein HSA-GLP-1 analysis in preclinical pharmacokinetics.

关 键 词：双抗体夹心ELISA HSA—GLP-1融合蛋白 药代动力学检测 

分 类 号：Q502[生物学—生物化学] R392.1[医药卫生—免疫学]