The Preparation Of B-type Natriuretic Peptide Monoclonal Antibody And The Study In Clinical Application

Objective:The early diagnosis of heart failure is very important , because the early treatment for the patients who were in trouble with the heart diseases is very important. As a member of the family of the natriuretic peptides, B-type natriuretic peptide(BNP) exists in many organs and tissues in the human body. Above all, BNP was secreted more from ventricular myocyte when ventricular loading is overdose than normal. The correlation between plasma concentration of BNP and cardiac function was well recognized. The detections of plasma BNP concentration were always use several kitts which were imported, the ranges and the cut off values were different, and it was more expensive for more application. To manufacture BNP kit ourself, this study was to prepare and characterize monoclonal antibodies(McAb) against BNP with high specificity and sensitivity, and detect the human plasma, evaluate the correlation between BNP concentration and cardiac function.Methods:A synthesized DNA segment coding for hBNP-32 was ligated to pET32 vector. The recombined plasmid was transfened into E. coli. The fusion protein were expressed by induction with IPTG and purified by Ni-NTA affinity chromatography in one step. DNA sequencing, SDS-PAGE and Western Blot were used to verify the plasmids and. Using the TRX-BNP fusion proteins to immunize BALB/C mouse with the standard technique. Performed ELISA screen to test the hybridomas' quality, limited dilution for cell cloning. The specificity and sensitivity of the BNP McAb were indicated by western blot and immunohistochemistry. Detect the human plasmaconcentration of BNP that with heart diseases (n=92) or healty (n=20) .The patients with heart diseases were divided to four teams which is on the basis of heart failure (Class I-IV). Evaluate the correlation between BNP concentration and cardiac function.Results:TRX-BNP fusion proteins were attained through prokaryotic expression method, contains immunogenicity and reactivity of antigen. After immunize BALB/C mouse with the standard technique, we found hybridomas which can steadly secret McAb against BNP. Western blotting indicated that BNP McAb specifically to BNP protein. Immunohistochemistry test showed positive staining for the sections of ventricular myocytes specifically. The results of the human plasma concentration of BNP that with heart diseases (n=92) or healty (n=20) show that, the BNP concentration were high in heart failure patients, and it is more high in Class IV than Class III, and by turns to Class II and Class I.Conclusion:BNP fusion protein was successfully constructed and the BNP McAb was prepared. The results of the human plasma concentration of BNP in healthy person and heart disease patients are concord with other results as known now. The worse heart function, the high BNP concentration.