Magnetic Nanoparticle-Based Automatic Chemiluminescent Enzyme Immunoassay For Golgi Protein 73 And The Clinical Assessment

ObjectiveLiver cirrhosis(LC),a common digestive systemic disease in the clinic,is a type of chronic,progressive,diffuse lesion of the liver caused by different causes.There is an increasing incidence in the recent years.However,in the early stage of the disease which is generally in the compensation period,there is no obvious clinical symptoms as well as the lack of specific examination methods.Thus,the patients easily miss the best diagnosis.In recent years,it has been found that Golgi protein 73(GP73)is mainly expressed in epithelial cells of various tissues in humans.It is not expressed in normal liver,but highly expressed in cirrhotic tissues.It is an independent Diagnostic indicators of cirrhosis.Its sensitivity in disease diagnosis is much higher than AFP,and it has become more and more popular as a potential new tumor marker.Almost all liver cancers have basal lesions of cirrhosis,so if liver cirrhosis can be diagnosed in time,it will undoubtedly play a certain early warning role in the diagnosis of liver cancer.However,there is currently no automated detection technology to cope with the clinical needs of large-scale medical examinations.Therefore,it is particularly important to be able to detect serum GP73 results more quickly and accurately.Methods1.The reactions in this paper was carried out on ACL2800 automated chemiluminescence analyzer.One kind of GP73 monoclonal antibody 8E7 labeled by biotin,and another kind of GP73 monoclonal antibody 5A10labeled by Acridinium ester(AE)were been used in this paper.The two monoclonal antibodies were complexed with GP73 antigen so to form a double antibody sandwich,and then combined with streptavidin modified magnetic beads.After cleaning with acid(HNO3+H2O2)and alkali(NaOH),the instantaneous chemiluminescence value is detected.The experiment determined the optimal working concentration and reaction conditions of various reagents in the method,and carried out clinical evaluation of the methodology.2.The serum levels of GP73 in 160 patients with cirrhosis and 50 healthy subjects were detected.The sensitivity and specificity of the patients were analyzed by ROC curve.The patients with cirrhosis were classified to Child-Pugh classification and their serum cholinesterase was detected.Cholinesterase,(CHE),Albumin(ALB),Total bilirubin(TBIL)and Prothrombin time(PT)levels were used to analyze the correlation between the above indicators and GP73.Results1.The automated magnetic enzyme-free GP73 chemiluminescence detection method was successfully established.The detection sensitivity of this method is 1.19 ng/mL,and the detection range is 1.34-684.38 ng/mL.The coefficient of variation(Coefficient of Variation,CV)obtained by precision analysis.CV is<2.69%,and the coefficient of variation between batches CV is<3.55%.The recovery rate is 93.46%to 107.82%.The method has a wide detection range,high sensitivity and specificity,and can meet the requirements for clinical detection of specimens.2.The level of serum GP73 in patients with liver cirrhosis was significantly higher than that in the normal control group(P<0.05).The area under the ROC curve was 0.886,with a cut-off value of 124.5 ng/mL.The sensitivity of GP73 in diagnosing cirrhosis was 78.05%.The specificity was 82.93%;serum GP73 levels in patients with cirrhosis were statistically different between Child-Pugh group(P<0.05)and correlated with cirrhosis indicators CHE,ALB,TBIL,PT(r=-0.690,r=-0.428,r=0.672,r=0.575,P<0.001).ConclusionThe above experiments show that this study has successfully established GP73’s fully automatic magnetic enzyme-free chemiluminescence quantitative detection method.This method provides a new reference for the detection of GP73,with high sensitivity and specificity.Moreover,the detection performance is superior over the existing enzyme-linked immunosorbent assay(ELISA).So that,this method provides a convenient tool for large-scale screening of medical personnel in the future.The level of serum GP73 increases significantly with the progression of cirrhosis and is associated with cirrhosis.It can be used as an effective indicator for the diagnosis and monitoring of liver cirrhosis.There is a important value in assessing the severity of cirrhosis by combining this method with the detection of GP73 and CHE,ALB,TBIL,and Pt.