| Objective:1Firstly,recombinant antigen of adiponectinwas constructed,and this antigen was used to producemonoclonal antibodies. Then adiponectin kit was established by monoclonal antibody which were screen out in previous study.2 To evaluate the value of adiponectin(ADPN) and amino-terminal pro-brain natriuretic peptide(NT-pro BNP) for the diagnosis of acute heart failure.Methods:1 6 His tags were introduced to N edgeof PTO-T7 vector aim to construct the cloning of adiponectin and insure it expressed in E.coli, then antigen were purified by metal chelating chromatography. So we use traditional monoclonal antibody technique to produce adiponectin monoclonal antibody. Double antibody sandwich method were used to select adiponectin antigen which could match successfully in ELISA kit. Finally quantitative ELISA kit was constructedby matched antibody.2 Clinical study enrolled 407 patients presenting with breathing difficulties,dyspnea, chest pain, chest oppression and othersymptoms of heart disease to the emergency department of Xiamen Cardiovascular hospitalbetween April and June 2015.218 patients were diagnosed with acute heart failure(AHF). All of participants completed NT-pro BNP, ADPN and other biochemical markers in hospital.(1) The AHF patients were classified into four groups based on NYHA cardiac functions and pathological causes, respectively. The difference of ADPN and NT-pro BNP levels in different groups was evaluated. In abnormal renal function group,the diagnostic value of ADPN(AUC=0.87,sensitivity 65.4% and specific 91.5%)(2) The correlations of ADPN and NT-pro BNP with renal function(GFR andcreatinine)were investigated and compared.(3) All participantswere divided into normal renal function group(GFR?60ml/min/1.73m2) and renal dysufunction group(GFR<60ml/min/1.73m2), receiver operating characteristic curve was used to anlyze the diangosticvalue of ADPN, NT-pro BNP, and the combination of ADPN and NT-pro BNP.Results:1 Adiponectin gene was amplified by PCR,the amplified products were passed by induction, expression, purification,thenactive fragment ADPN antigen were chosen to immunize mice.31 Hybridoma cell lines were selected to use quantitative ELISA kits because they could expressed adiponectin antigen stablely. At last m Ab-7B5 and 1E2-1 were chosen to developed quantitative ELISA kit.2 Both adiponectin and NT-pro BNP levels were significantly higher in participants with advanced NYHA classes.In plasma of adiponectin, p value of Class I and control group was 0.04, Class I and Class II was 0.116>0.05, Class II and Class III was 0.006,Class III and Class IV was 0.041. In plasma of NT-pro BNP, p value of Class I and control group was 0.262>0.05, Class I and Class II was 0.019, Class II and Class III was 0.054>0.05,Class III and Class IV was 0.024.The ADPN level was higher in no-ischemic HF patients than ischemic ones(p=0.023<0.05), but the NT-pro BNP levels hadno significant difference between no-ischemic HF patients and ischemic HF ones(p=0.81>0.05).3 The correlation of ADPN and renal function was weaker than correlation of NT-pro BNP and renal function.In control group, ADPN has no correlation with GFR and creatinine(p=0.213 and 0.121). R of NT-pro BNP and GFR,creatinine and were-0.488 and 0.386(p<0.05).In acute heart failure group, there were no correlation between ADPN and GFR or creatinine(p=0.164 and 0.288). R of NT-pro BNP and GFR and creatinine were-0.412 and 0.343.(p<0.05)4All subjects were divided in normal renal function group and abnormal renal function group by GFR=60 ml/min/1.73m2. In normal renal function group, NT-pro BNP were superior to ADPN(AUC=0.91 vs 0.76 the sensitivity was 93.1% vs43.9%and the specificity was 72.3% vs 94.9%).However, in the abnormal renal function group, ADPN(AUC=0.87 the sensitivity was 65.4% and the specificity was 91.5%)was superior to NT-pro BNP(AUC=0.83 the sensitivity was 90.4% and the specificity was 67.8%) for the diagnosis of AHF. But the differences had no statistical significance(p=0.34>0.05).5 Normal renal function group was divided into 3 groups by age(>75,50-75,<50), the best cutoff value of ADPN were 9.76ug/ml,8.21ug/ml and 8.76ug/ml. and the best cutoff value of NT-pro BNP were 1497pg/ml,335pg/ml and 221.5pg/ml.Abnormal renal function group were divided as described above, the best cutoff value of ADPN in above 75 years 11.98ug/ml,50-75 years 9.57 ug/ml and <50 years 7.89ug/ml,and the best cutoff value of NT-pro BNP were above 75 years 1129.5pg/ml,50-75 years 1219pg/ml and <50 years972pg/ml6Clinical Diagnostic Valueof the Combination: ADPN and NT-pro BNP were used to structurelogistic regression equation, and the predictor of equation was used to diagnosis acute heart failure. In normal renal function group(AUC=0.92 the sensitivity was 95% and the specificity was 75.9%),the combination of the two biomarkers did not have a higherdiagnostic efficiency than NT-pro BNP(p=0.12). In renal failure group, combined with the two biomarkers(AUC=0.90 the sensitivity was 71.2% and the specificity was 98.3%)could significantly increase the diagnostic efficiency than NT-pro BNP(p=0.016)and ADPN(p=0.024).Conclusions:1 Adiponectin ELISA kit was established by developing adiponectin antibody and applied in subsequent evaluation of clinical samples and they both increased in participants with advanced NYHA classes2 Thelevel of ADPN and NT-pro BNP increased in AHF patients and it hadpositive correlation with the seriousness of AHF.The correlation of ADPN and renal function was weaker than correlation of NT-pro BNP and renal function(GFR, creatinine, cystatin C). ADPN was less affected by renal fuctions and age in diagnosis AHF compared with NT-pro BNP.3 Compared with NT-pro BNP and ADPN as single diagnostic marker, combination of the two biomarkers could significantly increase the diagnostic efficiency. |
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