Establishment Of Test Method For Fucosyltransferase Of FUT4in Cancer Patients’ Serum And Its Clinical Significance

Objective: Tumor of glycosylation has a closely relationship with metastasisgrowth and prognosis of tumor. Fucosyltransferase (FUT) is a hexosyltransferase,according to fucosyltransferase catalytic types different, can be divided into13fucosyltransferase.A fucosyltransferase is an enzyme that transfers an L-fucose sugarfrom a GDP-fucose (guanosine diphosphate-fucose) donor substrate to an acceptorsubstrate. Esearchers showed that the FUT4complex is one of the new targets ofoncotherapy.To establish a rapid detection method for FUT4in human serum and applyit to early diagnosis of clinical tumor.Methods: Case selection:300cases of normal serum and370cases of tumorserum are selected. The tumor serums include gastric cancer, breast cancer, lung cancer,ovarian cancer, liver cancer, colon cancer. Detection FUT4content in serum by ELISA:and set the blank, negative, positive for comparison; add the serum to be tested;Blocking buffer add fut-4antibody; add IgG HRP antibody; add the substrate color; addthe Stop Solution;(1) Test of optimal concentration of FUT4:prepare solutionofglycosyltransferase FUT4with density1.500，1.1000,1.2000,1.3000,1.4000, test OD450and determine the optimal concentration.(2)Test of optimal concentration of IgG HRPantibody: prepare solution of HRP antibody:1:1000,1:2000,1:3000,1:4000,1:5000, testOD450and determine the optimal concentration.(3) The determination of comparisonpositive concentration: the cancer cell lysates of human endometrium cancer epithelialcells A431diluted into1:50,1:100,1:200,1:300,1:400five concentration, according tothe ELISA steps OD450value mapping, to determine the optimal concentration.(4)Test: use determined conditions to test the cancer patients’ serum FUT4OD450value.2.Express the fut-4results with Dot-ELISA method. Select NC membrane: add the serumto be tested; Blocking buffer add fut-4antibody; add IgG HRP antibody; add the DAB color; Use Western bolt method to analyze the FUT4expressing profile of the normalserum group and patient group.Results:1. Conditions determined in the ELISA: the serum concentration is90ul,the concentration of FUT4antibody is1:3000, and the concentration of positive controlA431cell lysate is1:200.Serum-coated condition is at4℃for overnight; FUT4response time is2hours; closed for an hour; color reaction time is20minutes.2.Results analysis: The correlations of all measurements were statistically analyzed bySPSS13.5. The FUT4expression levels of experimental group of cancer patients aresignificantly higher than the normal group with p <0.01. Therein the difference ofexpression level of gastric cancer, ovarian cancer, breast cancer, colon cancer, lungcancer, Liver cancer.3.Dot-ELISA shows that the tumor group is more obvious than inthe normal control group.4Western blot analysis also showed that the tumor band ismore apparent than normal band.Conclusion:1The ELISA method established in this research has characteristicsof sensitivity and specificity. It can be used to test the expression of the FUT4antigen inhuman serum. and normal serum FUT4expression level differences are moreobvious. The expressions of gastric cancer, ovarian cancer and breast cancer are moreobvious, which has significant meaning for the clinical diagnosis of stomach cancer.2. The trends of Dot-ELISA and Western blot experimental results areconsistent.The results show FUT4is a potential tumor marker which can be used forclinical preliminary the early detection of cancer.