【摘要】 目的:研究鉴定人脱嘌呤脱嘧啶核酸内切酶单克隆抗体(hAPE1 mAb)的抗原表位,并建立定量检测hAPE1的ELISA一步法。方法:设计并合成APE1-15肽阵列,鉴定hAPE1 mAb 2-G1和4-F6的抗原表位,应用三维立体结构观察软件Molsoft.ICM-Pro模拟hAPE1 mAb抗原表位的立体结构;采用改良的过碘酸钠法标记抗体,以hAPE1 mAb为捕获抗体和酶标抗体,建立hAPE1的ELISA一步检测法。结果:APE1-15肽阵列检测结果和抗原表位三维结构显示,2-G1mAb的抗原表位对应为hAPE1天然蛋白氨基酸残基序列的76-90位和109-123位,位于氧化还原区域,为构象型抗原表位;4-F6 mAb的抗原表位对应为hAPE1天然蛋白氨基酸序列的109-147位,位于DNA修复内切酶活性区。ELISA一步法检测hAPE1蛋白的线性范围为8.0~200μg/L,最低检测限为2.0μg/L。平均批内变异系数为8.67%,平均批间变异系数为12.45%,平均回收率为105.47%。结论:hAPE1 2-G1 mAb和4-F6 mAb具有不同的抗原表位,成功建立的hAPE1 ELISA一步法为简便、快速、准确检测血清中hAPE1的含量奠定了基础。更多还原

【Abstract】 AIM:To identify the antigenic epitopes of human apurinic/apyrimidinic endonuclease monoclonal antibodies(hAPE1 mAb) and to establish a one-step ELISA for quantitative measurement of hAPE1.METHODS:APE1-15 polypeptide array was used to identify the antigenic epitopes of hAPE1 mAb.Molsoft.ICM-Pro software was used to display the three dimensions of antigenic epitopes of hAPE1 mAbs.hAPE1 mAb was labeled with HRP by modified method of sodium periodate oxdization.A one-step ELISA was established by using two hAPE1 mAbs as a capture antibody and a detection antibody,respectively.RESULTS:The antigenic epitopes of 2-G1 mAb were localized within amino acids(aa) 76-90 and 109-123 in APE1 redox domain,belonging to the conformational epitope category.The antigenic epitope of 4-F6 mAb was within aa 109-147 in DNA repair endonuclease domain.In the range of 8.0 μg/L to 200 μg/L,the hAPE1 antigen showed a good linearity in the standard curve.The minimum detection threshold of this assay was 2.0 μg/L.The average of intra-assay precision and inter-assay precision were 8.67% and 12.45%,respectively.The average recovery rate was 105.47%.CONCLUSION:hAPE1 2-G1 mAb and 4-F6 mAb have different antigenic epitopes,and the one-step ELISA is established successfully to detect the serum hAPE1 level easily,quickly and accurately.更多还原