胰腺癌潜在肿瘤标志物的探索及在胰腺癌中的应用

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(P0.001)。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。

Detection of Potential Tumor Markers in Pancreatic Carcinoma and Its Application in Pancreatic Cancer

ABSTRACT Objective: To find the potential tumor marker of pancreatic cancer and establish its sandwich ELISA system, and apply it to the detection of the serum of patients with pancreatic cancer. Methods: MALDI-TOF-MS was used to analyze the serum of preoperative and postoperative pancreatic cancer patients. The DAP44 protein was purified and purified by hybridoma technique. The anti-DAP44 monoclonal antibody was prepared by hybridoma technique. The antibody titer was detected by HRP labeling method. Antibody titer was detected by indirect ELISA. Histopathological staining of pancreatic cancer tissue and adjacent tissue was performed with the prepared antibodies. Anti-DAP44 kit was prepared by sandwich ELISA (DAS-ELISA) Pancreatic cancer patients and normal serum DAP44 values, compared the difference. Results: The differential protein peaks of preoperative and postoperative pancreatic cancer were detected and collected. Peptide sequencing and bioinformatics analysis were performed on the differentially expressed proteins. Three hybridoma cells stably secreting monoclonal antibodies against DAP44 (2D6H5, 1E4D6, 5B8H12 , 3) hybridoma cells secreted antibody titers above 107, by antibody matching screening to determine 2D6H5 as coating antibodies, 1E4D6 for the enzyme labeled antibody. The results of histochemical staining showed that the expression of DAP44 in cancer tissues was much higher than that in adjacent tissues. The linear range of DAS-ELISA was 0.78-25ng / ml and 0.78ng / ml, respectively. The detection of DAP44 in pancreatic cancer and The average content of DAP44 in normal group was 19.707 ± 1.464 and 10.653 ± 2.221, respectively. There was significant difference between the two groups (P <0.001). Conclusion: DAP44 could be used as a potential marker for the pancreatic cancer. The established anti-DAP44 DAS-ELISA system could be used for the clinical diagnosis and assessment of curative effect of pancreatic cancer.