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目的通过制备的人组织型激肽释放酶6(hK6)单克隆抗体(mAb),建立双抗夹心酶联免疫吸附测定(ELISA)检测方法,并用于胃癌诊断。方法采用该实验室保存的hK6重组蛋白,免疫BALB/c小鼠后通过杂交瘤技术制备特异性的mAb,经鉴定纯化、酶标记后,建立双抗夹心ELISA方法,检测胃癌患者血清中hK6水平,并联合检测血清中的癌胚抗原(CEA)水平,探讨hK6作为胃癌生物标记的可行性。结果成功建立了检测血清中hK6的双抗夹心ELISA方法,并确定该方法包被抗体的最适浓度为5μg/mL,酶标记抗体的最佳稀释比例为1∶2 000。该方法检测各组血清中的hK6水平,与胃溃疡组hK6水平[(3.59±1.02)ng/mL]和健康体检组hK6水平[(3.35±0.67)ng/mL]比较,胃癌组hK6水平[(5.78±1.66)ng/mL]明显高于其他两组,差异有统计学意义(P<0.05);而胃溃疡组与健康体检组hK6水平比较,差异无统计学意义(P>0.05)。胃癌患者hK6和CEA检测的阳性率分别为69.70%和45.46%,两者联合检测的阳性率为78.79%。结论成功建立了hK6双抗夹心ELISA检测方法;hK6是较好的胃癌血清肿瘤标记物,同时检测hK6与CEA可提高胃癌的检出率,减少漏诊的发生,有助于胃癌的诊断。
OBJECTIVE To establish a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the diagnosis of gastric cancer by preparing human monoclonal kallikrein 6 (mAb). Methods The recombinant hK6 protein was isolated from BALB / c mice and the specific mAbs were prepared by the hybridoma technique. The purified mAbs were identified by enzyme-linked immunosorbent assay (ELISA), and the serum hK6 level , And combined detection of serum carcinoembryonic antigen (CEA) levels, to explore the feasibility of hK6 as a gastric cancer biomarker. Results A double-antibody sandwich ELISA method for detecting hK6 in serum was successfully established. The optimum concentration of coated antibody for hK6 was 5 μg / mL and the best dilution ratio of enzyme-labeled antibody was 1: 2,000. HK6 levels in gastric ulcer group [(3.59 ± 1.02) ng / mL] and hK6 level in healthy control group [(3.35 ± 0.67) ng / mL], hK6 levels in gastric cancer group [ (5.78 ± 1.66) ng / mL] was significantly higher than the other two groups (P <0.05). There was no significant difference in hK6 levels between gastric ulcer group and healthy group (P> 0.05). The positive rates of hK6 and CEA in gastric cancer patients were 69.70% and 45.46%, respectively. The positive rates of hK6 and CEA in gastric cancer were 78.79%. Conclusion hK6 double antibody sandwich ELISA has been successfully established. HK6 is a good marker of gastric cancer serum. Simultaneous detection of hK6 and CEA can improve the detection rate of gastric cancer, reduce the missed diagnosis and contribute to the diagnosis of gastric cancer.