Establishment Of REGⅠα Time-Resolved Fluoroimmunoassay And Its Clinical Application In Gastric Cancer

Background: Regenerating protein I alpha(REGⅠα)is a gastric mucosal cell growth factor produced by enterochromaffin-like(ECL)cells and chief cells of human gastric mucosa,which plays an important role in the proliferation,differentiation and apoptosis of gastric mucosal cells.A growing body of evidence showed that REGⅠαm RNA and protein were highly expressed in GC specimens and were correlated with infiltrative characteristics,venous invasion,tumor stage,differentiation type,lymph node metastasis,and poor prognosis in patients with GC.However,studies on REGⅠαin the serum of patients with GC are scarce,and the correlation between REGⅠα serum levels and the clinicopathological characteristics of patients with GC remains to be elucidated.Therefore,the aim of this study was to establish a highly sensitive timeresolved fluoroimmunoassay(TRFIA)for the quantitative analysis of human serum REGⅠα levels and to investigate the clinical application value of REGⅠα in GC patients from a serological perspective.Methods: In this study,two monoclonal antibodies specific for REGⅠα with different epitopes of antigen molecules were used as the capture antibody and the detection antibody,respectively.A REGⅠα time-resolved fluorescence immunoassay(REGⅠα-TRFIA)was developed for the quantitative determination of REGⅠα levels in human serum by solid-phase solidification of capture antibodies,labeling the detection antibodies with lanthanide Europium(Eu)as a tracer,and evaluating its analytical performance using the reaction mode of double antibody sandwich method.The established REGⅠα-TRFIA method was used to quantify the serum levels of REGⅠα in148 healthy controls(HCs),69 patients with benign gastric disease(BGD)and 125 patients with GC,and to analyze the clinical value of serum REGⅠα in the auxiliary diagnosis and prediction of disease progression in GC,taking into account the clinicopathological characteristics of the patients.Results: 1.The REGⅠα-TRFIA method had a limit of quantification of 0.94 ng/m L with an optimal reaction system and a detection range of 0.94 ng/m L-1000 ng/m L.When the concentration of REGⅠα in the sample was less than 10,000 ng/m L,no hook effect was observed with the REGⅠα-TRFIA method.The intra-assay coefficients of variation ranged from 2.01%-2.94% and the inter-assay coefficients of variation ranged from 4.23%-4.77%.The ratio of measured to theoretical concentrations in the recovery experiments ranged from 90.33%-100.85%,which was within the acceptable range of90-110%.Triglycerides(10 mmol/L),bilirubin(350 μmol/L)and hemoglobin(10 g/L)did not interfere with the performance.The results of REGⅠα-TRFIA and a commercial enzyme-linked immunosorbent assay showed good correlation and consistency(ρ =0.985).2.Serum REGⅠα levels were 110.64(89.14-141.58)ng/m L in the HCs group,133.99(104.26-187.85)ng/m L in the BGD patients,and 262.28(183.77-387.98)ng/m L in the GC patients.Serum REGⅠα levels were higher in patients with BGD than in HCs,with a statistically significant difference(P = 0.0005).Serum REGⅠα levels were significantly higher in GC patients than in HCs and BGD patients,with statistically significant differences(P < 0.0001).3.Serum REGⅠα levels were significantly higher in GC patients with TNM stage(III-IV)than in GC patients with TNM stage(I-II),with a statistically significant difference(P = 0.0044).Serum REGⅠα levels were significantly higher in GC patients with tumor invasion depth(T3-T4)than in GC patients with tumor invasion depth(T1-T2)(P = 0.0008).Serum REGⅠα levels were higher in GC patients with distant metastases than in GC patients without distant metastases(P = 0.0068).When comparing serum REGⅠα levels in GC patients with or without lymph node metastases,there was no significant difference in REGⅠα levels(P > 0.05).Serum REGⅠα levels were higher in GC patients with poorly pathological differentiation type than in GC patients with moderately to highly pathological differentiation type(P = 0.0257).4.The diagnostic efficiency of the combined REGⅠα,carcinoembryonic antigen and carbohydrate antigen199 assay(area under the curve [AUC] value: 0.9561,sensitivity: 88.00%,specificity: 93.24%)for GC was significantly better than that of the REGⅠα(AUC value: 0.9274,sensitivity: 84.00%,specificity: 88.51%),CEA(AUC value: 0.7282,sensitivity: 66.40%,specificity: 68.24%)and CA199(AUC value:0.6963,sensitivity: 38.40%,specificity: 93.24%)assay individually by receiver operating characteristic(ROC)curves analysis.Conclusions: 1.In this study,the REGⅠα-TRFIA method was successfully established,and all performance indicators of the method meet the requirements of clinical testing reagents.2.Serum REGⅠα levels were slightly elevated in patients with BGD and significantly elevated in patients with GC,which indicated that elevated serum REGⅠαlevels may be closely related to the occurrence of BGD and further development of GC,and that REGⅠα may be a biomarker for the ancillary diagnosis of BGD and GC.3.The serum level of REGⅠα in GC patients significantly increased with more advanced TNM stage,poorer pathological differentiation type,deeper tumor invasion depth(T3-T4)and patients with distant metastasis,and the high level of REGⅠα in the serum could reflect the development and deterioration of GC to a certain extent.4.The combination of serum REGⅠα with CEA and CA199 could effectively improve the clinical diagnosis of GC.