【摘要】 建立一种非抗体依赖的检测外源性蛋白多肽分子代谢动力学血清浓度及动态变化的新方法.链亲和素为捕获分子,加入待测生物素标记蛋白或合成肽,辣根过氧化物酶标记链亲和素为检测分子,构成链亲和素-生物素标记大分子-酶链亲和素的双夹心体系,并进行特异性、敏感性、准确性及稳定性评价.本检测体系灵敏度高,可达0.3125μg/L,且可通过改变亲和素包被浓度调整检出敏感度与检测范围.准确性回收率为97.82%～107.92%,批内、批间变异系数分别<5.76%和<8.42%,并成功应用在生物素标记人血清白蛋白(biotin-HSA)与生物素标记鸡卵黏蛋白(biotin-OVM)的小鼠血清浓度动态检测.本方法不依赖抗体与放射性核素,可望在外源性蛋白多肽大分子及其药物的体内定量检测和代谢动力学研究中广泛应用.更多还原

【Abstract】 For the assessment of the dynamic concentration of exogenous protein or peptide in serum,an antibody-independent sandwich system based on the avidin-biotin interaction was established and validated.The exogenous protein or polypeptide labeled with biotin was added to the microplate coated with streptavidin,and followed by adding HRP-streptavidin to complete the sandwich system.This sandwich system was validated with respect to specificity,sensitivity,accuracy(recovery) and reproducibility.While the sensitivity of detection could reach to 0.3125 μg/L,the sensitivity and the range of detection can also be adjusted by changing coating concentration of streptavidin.Recoveries ranged from 97.82% to 107.29%,and the intra-and inter-assay variation was < 5.76% and < 8.42%,respectively.Thus,the well-validated streptavidin sandwich system was successfully applied to determine dynamic concentrations of Biotin-HAS(human serum albumin) and Biotin-OVM(Ovomucoid) in mouse serum,respectively.Most importantly,this novel method,where generation of antibody or radionuclide is not needed,may provide a new choice for assessment or monitoring of exogenous protein or peptide in pharmacokinetics study.更多还原