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目的:探索胰腺癌新的潜在标志物,建立夹心法ELISA体系,并初步应用于胰腺癌患者的血清检测。方法:应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术对胰腺癌患者术前后血清进行分析,提纯分析差异蛋白并命名为DAP44,通过杂交瘤技术制备出抗DAP44单克隆抗体,用HRP标记法标记抗体,间接ELISA法检测抗体滴度,用制备出的抗体对胰腺癌组织和癌旁组织进行组化染色,采用夹心ELISA(DAS-ELISA)法制备抗DAP44检测试剂盒,检测胰腺癌病人和正常人血清DAP44值,比较两者差异。结果:对差异蛋白进行肽段测序和生物信息分析,并融合了3株能稳定分泌抗DAP44单克隆抗体的杂交瘤细胞(2D6H5,1E4D6,5B8H12),3株杂交瘤细胞分泌的抗体效价均在107以上,通过抗体配对筛选确定以2D6H5为包被抗体,1E4D6为酶标抗体时,DAS-ELISA法敏感性最高。两株抗体组化染色结果显示:癌组织DAP44表达量远高于癌旁。DAS-ELISA法标准曲线线性范围在0.78-25 ng/mL,检测下线为0.78 ng/mL,此方法检测到的胰腺癌病人和正常人血清DAP44平均含量分别为19.707±1.464和10.653±2.221,两者之间有统计学差异(P<0.001)。结论:DAP44可能作为潜在的胰腺癌肿瘤标志物,建立的抗DAP44 DAS-ELISA法体系能够初步用于胰腺癌的临床诊断和疗效评估指标。
OBJECTIVE: To explore new potential markers of pancreatic cancer and to establish a sandwich ELISA system for the preliminary detection of serum in patients with pancreatic cancer. Methods: Serum samples of patients with pancreatic cancer were analyzed by matrix-assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF-MS). The differential proteins were purified and named as DAP44. Anti-DAP44 monoclonal antibodies , The antibody was labeled with HRP, the antibody titers were detected by indirect ELISA, the pancreatic cancer tissues and adjacent tissues were stained with the prepared antibodies, and the anti-DAP44 kit was prepared by sandwich ELISA (DAS-ELISA) Serum DAP44 levels were measured in patients with pancreatic cancer and normal controls, and the differences between the two were compared. Results: Three different hybridoma cells (2D6H5, 1E4D6 and 5B8H12) that can stably secrete anti-DAP44 monoclonal antibody were fused by peptide sequencing and bioinformatics analysis. The antibody titers secreted by the three hybridoma cells were Above 107, DAS-ELISA was the most sensitive when using 2D6H5 as the coated antibody and 1E4D6 as the ELISA antibody by paired antibody screening. The results of two antibody histochemical staining showed that the expression of DAP44 in cancer tissues was much higher than that in adjacent tissues. The standard curve of DAS-ELISA showed a linear range of 0.78-25 ng / mL, and the lower limit of detection was 0.78 ng / mL. The mean DAP44 levels in serum of patients with pancreatic cancer detected by this method were 19.707 ± 1.464 and 10.653 ± 2.221, respectively, There was a statistically significant difference between the two (P <0.001). Conclusion: DAP44 may serve as a potential tumor marker for pancreatic cancer. The established anti-DAP44 DAS-ELISA system can be used primarily in the clinical diagnosis and therapeutic evaluation of pancreatic cancer.