Construction Of SSt2 Double Antibody Sandwich ELISA Kit And The Application In Heart Failure

OBJECTIVE:To construct ST2 recombinant protein for preparing monoclonal antibody,and then establish a double antibody sandwich ELISA kit for quantitative detection of sST2 in clinically.And then assess the diagnostic value of sST2 in heart failure and compare the correlation between sST2,NT-proBNP and renal function.METHOD:1.The human sST2 gene was amplified by PCR and the target fragment was ligated with the PET-30a vector to construct the recombinant plasmid.The recombinant plasmid was transferred to E.coli for expression and then the protein was purified by Mickey ion affinity column.And then immunized with BALBc mice with sST2 high affinity specific monoclonal antibody.2.Construct sST2 double antibody sandwich ELISA kit by using biotin avidin system.The construction of the kit includes screening of antibody pairings,selection of reaction systems,determination of the concentration of biotin-labeled antibody and the enzyme concentration,determination of standard curve,linear range,correlation evaluation,recovery rate test and precision.3.Clinical samples were analyzed by using sST2 double antibody sandwich kit.The diagnostic value of sST2 in heart failure was analyzed by ROC curve.Compare the relevance of sST2,NT-proBNP and creatinine and glomerular filtration rate(GFR).RESULT:1.The recombinant plasmid was successfully constructed and transferred into E.coli for expression.The purified sST2 antigen was obtained by protein purification,and the mice were immunized with it.At last,64 specific monoclonal antibodies with high affinity for sST2 were successfully screened.2.14G5(capture antibody)and 15G3(detectable antibody)were successfully screened an antibody pairs for sST2 double sandwich ELISA kit.The reaction system and reaction conditions were follows:using ED-11 system as the best reaction system;the working concentration of the biotin-labeled antibody was 1:250;the concentration of HRP was 1:3000;The linearity of the standard curve is:y = 0.081x + 0.019,R2=1,the linear range is 0.4～40 ng/mL;with 40 clinical samples and R&D kit parallel test,Spearman’ s coefficient of rank correlation:0.825(95%CI:0.691-0.904;P<0.001),the results show that the both have a good correlation;The average recovery is 94.99%and the kit is well tested;intra-assay and inter-assay CV were less than 7%show that the kit reproducibility is good.3.The area under the ROC curve of sST2 is 0.842.When the cutoff value of sST2 is set to 3.275ng/mL,the sensitivity is 74.15%and the specificity is 82.05%.The level of sST2 in the heart failure group was higher than that in the control group,the difference was statistically significant(P<0.001).There was no correlation between sST2 and creatinine and GFR in patients with heart failure.The values of P were 0.822 and 0.081 respectively.NT-proBNP was correlated with creatinine and GFR.The R values were 0.406 and-0.487(P<0.001),respectively.CONCULUSION:1.sST2 recombinant protein was successfully prepared by prokaryotic expression system,and sST2 monoclonal antibody was successfully prepared by monoclonal antibody preparation technique.2.we selected a pair of the best pair of monoclonal antibodies for sST2 detection.The optimum response system of this antibody pair was ED-11 system,and the biotin-labeled antibody and SA-HRP concentration were selected.The sST2 double antibody sandwich ELISA kit was successfully constructed.It can be used in the clinical detection of sST2 and make up for the gaps in the domestic market.3.sST2 can be used for the diagnosis of heart failure in clinically.The diagnostic value of sST2 is not affected by renal function in heart failure patients,to a certain extent,better than NT-proBNP.