CAJ | 학술논문

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。
목적：：위검측혈청중HSA-GLP-1융합단백정체분자적농도，건립일충특이、령민적정량검측식해후체내HSA-GLP-1융합단백농도적쌍항체협심ELISA적방법。방법：채용쌍항체협심ELISA방법，이GLP-1단극륭항체위포피항체、HSA-GLP-1융합단백위협심항원、anti-HSA-Biotin위검측항체，용Streptavidin-HRP진행면역방대，TMB현색。결과：건립료검측HSA-GLP-1융합단백적ELISA방법，기선성범위위15.6~1000 ng/mL，최저검측한위15.6 ng/mL，여GLP-1、HSA、IL2-HSA균무교차반응，판내화판간정밀도균소우15%，준학도위±15%，동융은정성화희석은정성량호。결론：건립적HSA-GLP-1단백검측방법부합신생물제품림상전약대동역학연구지도원칙요구，가용우HSA-GLP-1융합단백재림상전약대동역학시험적정량검측。
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.