| Objective: Chronic Congestive heart failure(CHF Congestive heart failure) is the end stage in the progress of various heart disease, which had seriously affected the health of patients. Thus, how to diagnosis and guide clinicians treatment timely in the early stage of heart failure has been a hot research. It had found that the change of N terminal forebrain sodium peptide(NT-pro BNP) content is the key marker to diagnose the degree of heart failure, which could reflect the variation of ventricular function and the damage of heart function sensitivity and specifically. At present, the research on the detection of NT-pro BNP is still in the initial stage in China. Most of the materials, including the kit antibody dependent on imports, which had been hinder the development of immunology and the elevation of technology in China. On the base of produce anti NT- pro BNP monoclonal antibody, the study intends to establish a double antibody sandwich ELISA detection method used for detect the content of NT–pro BNP in blood serum.Methods: The study use the NT-pro BNP antigen to immunize BABL/c mice, after fusion, screening and prepare of monoclonal antibody against NT-pro BNP. In the study, apply linear fragment 18~38, 35~50, 55~72 coupling with OVA respectively, to ensure linear epitopes were against by monoclonal antibody. We acquired paired monoclonal antibodies by double antibody sandwich ELISA method, and then the coated and blocked conditions, optimal antibodies working concentrations, reaction mode, linear range, assay precision, recovery rates, interference factors and clinical serum detection were studied to establish a quantitative ELISA method of NT pro-BNP and analyze its performance.Results: We finally obtain a total of 15 strains of monoclonal antibodies against NT-pro BNP. By epitope identification, there are five strains against 18~38 linear epitopes, two strains against 35~50 linear epitopes, three strains against 55~72 linear epitopes. By specificity identification, ten strains of monoclonal antibodies only antigen-reactive against NT-pro BNP, and no cross reaction with heart failure related markers, antibody pairs for 5E10 and 5G5. We explore a optimal detection conditions, it is the working concentration of coated antibody was 4μg/m L(diluted with 0.05 mol/L p H 9.6 CB), the working concentration of HRP-conjugate reagents were 1:3000, the samples were incubated for 30 min at 37℃, then the HRP-conjugate reagents were incubated for 30 min at 37℃ and evaded the light preservation to color for 20 min at 37℃. The linear range of method was 50-500 pg/m L to detect NT-pro BNP, Intra-assay Coefficients of variation(CV) were 2.3%, inter-assay CVs were 3.4%. The recovery rates of value serum were 101.2%(High), 97.2%(mid), 93.7%(low), respectively. The interference factors, such as contain jaundice, blood fat and lower hemoglobin did not have effects to results. During the test of 200 patients serum used for clinical application, the results suggest that the overall coincidence rate was 96.9%. Compared with abroad kit that detecting 48 patients serum simultaneously, the correlation coefficient was R2=0.9027.Conclusion: We successfully obtain ten strains NT-pro BNP by using monoclonal antibody, and get a pair of antibody to detection NT-pro BNP. A quantitative ELISA method for detection NT pro-BNP contents was preliminarily developed, which showed high accuracy and precision, and can provide timely and accurately basis for clinical. |
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