Preparation Of Monoclonal Antibody Against Human NT-proBNP

NT-proBNP is a linear polypeptide fragment secretded by cardic muscle cells during thesynthesis of peptide hormone BNP and has no bioactivity. It includes76amino acids and itsrelative molecular mass is about8.5KD. In the peripheral blood of healty people, theconcentration of NT-proBNP is very low and less then450pg/ml usually. When capacity loadcausing the change of ventricular pressure and the increase of wall tension, ventricular musclecells will synthetize and secrete BNP and NT-proBNP. Consequently the rise of NT-proBNP’sconcentration can well reflect the change of ventricular structure and function. Nowadays,NT-proBNP has been regconized as a sensitive and specific index of cardiac dysfunction anda objective marker having epoch-making significance in the diagnosis of heart failture.In this text, recombinant protein with label named hrNT-proBNP-DSBA was used asimmunogen to immunize Balb/c mice and recombinant protein with no label namedhrNT-proBNP was used as testing antigen for screening. When the titers of the mice’s tailserum reach the requestion for cell fusion, the mice’s spleen cells were harvested and fusedwith SP2/0myeloma cells. After enzyme-linked immunosorbent assay(ELISA) screening andsubcloning by limiting dilation,4stable hybridoma cell lines which can secreteanti-NT-proBNP monoclonal antibodies(mAbs) were establish. Ascites were produced byinjecting cells to mice’s abdomen and monoclonal antibodies were purified from ascites bycaprylic acid-ammonium sulfate precipitation method. And then, basic functions of thesemonoclonal antibodies, such as subclasses, purities, titers, specificity, affinity constants andepitopes, were analysed.7strong positive pairs of monoclonal antibodies were screened outby sandwich ELISA and were applied in fluorescent latex immunochromatographic platformfor the second time screening. Finally, an antibody pair consisting of capture(mAb1G12) anddetector(mAb H3) that showed a relatively higher sensitivity and clinical coincidence ratewere selected. The result of clinical verification shows that, in the fluorescent lateximmunochromatographic platform, the clinical relative coefficient R2(100serum samples) ofthe antibody pair consisting of1G12and H3is0.9451, negative coincidence rate is94.1%,positive coincidence rate is89.8%and the total coincidence rate is92%. This study has laid afirm foundation for development of human NT-proBNP quantitative rapid diagnostic reagentsfor clinical application.