摘要
目的：通过抗体配对方法，建立能高特异、高灵敏地定量检测食蟹猴体内IL-2-HSA融合蛋白浓度的双抗体夹心ELISA法。方法：以IL-2单克隆抗体为包被抗体、IL-2-HSA融合蛋白为夹心抗原、生物素标记的HSA为检测抗体，一抗和二抗的工作浓度分别为8Ixg／mL和1：5000，HRP标记的亲和素为1：200。结果：IL-2-HSA融合蛋白标准品的曲线范围为3．9-250ng／mL，最低检测限为3．9ng／mL，-9IL-2、HSA、GLP-1／HSA和CD20单抗均无交叉反应，方法的回收率为98．9％~101．5％，批内和批间准确度分别为96．1％～98．3％和93．9％～105．4％。结论：本方法符合新生物制品临床前药代动力学研究指导原则的要求，可用于IL-2-HSA融合蛋白在临床前药代动力学试验的定量检测。
Objective： To develop a high specific, high sensitive double antibody sandwich ELISA method tor the quantification of fusion protein IL-2-HSA by method of antibody pairing. Methods： Using an anti human IL-2 monoclonal antibody for capture was 8 ~g/mL, and a biotin-labeled of another anti-HSA monoelonal antibody for detection antibody was 1：5000. HRP labeled conjugate streptavidin was 1：200. Results： The standard curve was developed with a wide dynamic range of concentrations from 3,9 to 250 ng/mL. The lowest quantification of this assay was 3.9 ng/mL. The specificity assay indicated that it had no cross-reaction with IL-2, HSA, GLP-1/HSA and CD20. The recovery of method was 98.9%-101.5%, and the accuracy of the intra- and inter-assay were 96.1%-98.3% and 93.9%-105.4% respectively. Conclusion： The assay is highly sensitive, accurate, specific, and reproducibility over a wide dynamic range of concentrations, which was proven to be a feasible quantitative meth- od for fusion protein IL-2-HSA analysis in preclinical pharmacokineties.
出处
《生物技术通讯》
CAS
2013年第2期214-218,共5页
Letters in Biotechnology
