Development Of ELISA Kit For Detecting Serum Anti-CDC25B Autoantibody And Its Application In The Patients With Esophageal Squamous Cell Carcinoma

BACKGROUNDThe oncogene CDC25B is one member of CDC25 phosphatase family,it plays an important role in cancer cell growth. It has been recently reported that serum levels of CDC25B autoantibodies (CDC25B-Abs) in patients with esophageal squamous cell carcinoma (ESCC) is significantly higher than both healthy individuals and patients with other types of cancer。However, the potential diagnostic or prognostic significance of CDC25B-Abs in patients with ESCC is not clear. The aim of this study is to develop the reverse capture enzyme-linked immunosorbent assay (ELISA) kit and to detect the serum CDC25B–Abs of the ESCC patients by the ELISA kit in order to evaluate the clinical significance of CDC25B-Abs in patients with ESCC.METHODSAfter lysised ESCC Eca-109 tumor cells ,protein as antigen were purified to delvelop the reverse capture ELISA kit. CDC25B Abs was measured in serum from both 134 patients with primary ESCC and 134 healthy controls by a reverse capture ELISA in which anti-CDC25B antibodies bound CDC25B antigen purified from Eca-109 ESCC tumor cells. The significance of the measuring serum CDC25B Abs in the pateins with ESCC was compared with the measuring other tumor markers carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCC-Ag).RESULTSThe serum CDC25B–Abs of the ESCC paeints was able to be detected by the reverse capture ELISA kit, antigen purified from Eca-109 ESCC tumor cells. Higher levels of CDC25B -Abs were found in serum from patients with ESCC (A450 =0.9173±0.4729) than in serum from healthy control subjects (A450=0.3775±0.2618, P < 0.01). The area under the receiver operating characteristic (ROC) curve for CDC25B-Abs was 0.870 (95% CI: 0.835-0.920). The sensitivity and specificity of CDC25B-Abs for detection of ESCC were 56.7% and 91.0%, respectively, when CDC25-Abs-positive samples were defined as those with an A450 greater than the cut-off value of 0.7254. Relatively few patients tested positive for the tumor markers CEA and SCC-Ag (13.4% and 17.2%, respectively). A significantly higher number of patients with ESCC tested positive for a combination of CEA, SCC, and CDC25B-Abs (60.4%) than for a combination of CEA and SCC-Ag (26.9%, P < 0.0001). Although examination of the total patient pool showed no obvious relationship between CDC25B Abs and overall survival。The concentration of CDC25B Abs in serum was significantly correlated with tumor stage (P < 0.01)。In the subgroup of patients with stage III-IV tumors the cumulative five-year survival rate of CDC25B-seropositive patients was 6.7%, while that of CDC25B-seronegative patients was 43.4% (P = 0.001, log-rank). In the N1 subgroup, the cumulative five-year survival rate of CDC25B-seropositive patients was 13.6%, while that of CDC25B - seronegative patients was 54.5% (P = 0.040, log-rank).CONCLUSIONS1. The serum CDC25B–Abs of the ESCC patients may be detected by the reverse capture ELISA kit, whose antigen is purified from the protein of ESCC Eca-109 tumor cell .2. Serum CDC25B-Abs is superior to other tumor markers CEA and SCC-Ag for diagnosis of ESCC;3. CDC25B-Abs is a potential prognostic serological marker for advanced ESCC.