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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。
OBJECTIVE: To study the identification of human epitope of human apurinic aptamer endonuclease (hAPE1 mAb) and establish a one-step ELISA for the quantitative detection of hAPE1. Methods: The APE1-15 peptide array was designed and synthesized to identify the epitopes of hAPE1 mAb 2-G1 and 4-F6. The three-dimensional structure observation software Molsoft was used to simulate the three-dimensional structure of hAPE1 mAb epitope. Antibody was labeled with sodium periodate, and the hAPE1 mAb was used as the capture antibody and enzyme-labeled antibody to establish a one-step ELISA assay of hAPE1. Results: The results of APE1-15 peptide array and the three-dimensional structure of the epitope showed that the epitopes of 2-G1 mAb corresponded to 76-90 and 109-123 of the amino acid residues of hAPE1 native protein, located in the redox region Conformational epitope; 4-F6 mAb epitope corresponding to the hAPE1 amino acid sequence of amino acids 109-147, located in the DNA repair endonuclease activity. The linear range of hAPE1 protein was 8.0 ~ 200μg / L by ELISA and the lowest detection limit was 2.0μg / L. The average intra-assay CV was 8.67%, the average inter-assay CV was 12.45%, and the average recovery was 105.47%. CONCLUSION: The hAPE1 2-G1 mAb and 4-F6 mAb have different epitopes. The successful one-step hAPE1 ELISA established the basis for the simple, rapid and accurate detection of hAPE1 in serum.