Objective To prepare the mAbs against hK6 for establishing a sandwich enzyme-linked immunosorbent assay(ELISA) of hK6,and exploring its clinical value.Methods The hybridoma technique was used to prepare mAbs against hK6.The mAbs were purified and labelled with horseradish peroxidase for the sandwich ELISA method.The sandwich ELISA method was used to detect the serum hK6 concentrations in patients with malignant gastric neoplasm.Then the best antibody pair was selected from coating antibody and enzyme-linked antibody to establish a sandwich ELISA method through the chessboard titrations.Compared with CEA,we explored the feasibility of hK6 as gastric cancer biomarkers.Results A sandwich ELISA method was established for quantifying hK6 in serum.The results showed that the optimal concentration of coating antibody was 5 μg/mL.The optimal concentration of enzyme-linked antibody was 1:2 000.Serum hK6 in the patients with gastric cancer groups[(5.78±1.66)ng/mL] than healthy individuals groups[(3.35±0.67)ng/mL] and those in gastric ulcer groups[(3.59±1.02)ng/mL],the difference was statistically significant(P<0.05).Furthermore,there was no significant difference in values of serum hK6 between patients with gastric ulcer groups and healthy individuals groups(P>0.05).The hK6 positive rate of gastric cancer was 69.70％,and CEA was 45.46％.In the combined detection,the positive rate was 78.79％.Conclusion A sandwich ELISA is established successfully.As a favorable serum biomarker for gastric cancer,the detection of hK6 together with CEA is helpful in the diagnosis of gastric cancer.%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5 μg/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.
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