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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(P0.05).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.
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AIM OF THE STUDY: This study aims to investigate the expression of human kallikrein 6 (hK6) in gastric cancer, gastric ulcer and normal gastric mucosa tissues and its biological significance. MATERIAL AND METHODS: The expression of hK6 in 15 normal gastric mucosa (NGM) tissues, 15 gastric ulcer (GU) tissues and 55 gastric carcinoma (GC) tissues was respectively detected by immunohistochemistry. The correlations between the expression of hK6 and the clinical pathological parameters of gastric cancer were also analyzed. RESULTS: Human kallikrein 6 was mainly expressed in cytoplasm. The positive rate of hK6 was significantly higher in gastric cancer tissues than that in gastric ulcer or normal gastric mucosa tissues (70.9%, 40% and 20%, respectively, p < 0.01). With the increase of the invasion depth of gastric cancer cells, aggravation of TNM stage and development of lymph node metastasis, the expression of hK6 increased significantly (p < 0.05 and p < 0.01). There was no obvious correlation between the expression of hK6 and sex, age, tumor diameter, histodifferentiation degree or primary pathological location of gastric cancer (p > 0.05). CONCLUSIONS: The overexpression of hK6 is related to the depth of invasion, lymph node metastasis and clinical stage of gastric carcinoma, which suggests that hK6 may act as a new marker of gastric cancer biological behavior.
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A series of compounds with an amidinothiophene P1 group and a pyrrolidinone-sulphonamide scaffold linker was identified as potent inhibitors of human kallikrein 6 by structure-based virtual screening based on the union accessible binding space of serine proteases. As the first series of potent nonmechanism-based hK6 inhibitors, they may be used as tool compounds for target validation. An X-ray structure of a representative compound complexed with hK6, resolved at a resolution of 1.88 Å, revealed that the amidinothiophene moiety bound in the S1 pocket and the pyrrolidinone-sulphonamide linker projected the aromatic tail into the S' pocket.
