Preparation Of Monoclonal Antibodies Against Human Natriuretic Peptide

Background: Heart failure is the fourth cause of disease in adult hospitalization, which brings great threat to the human health. Since the peptide of Brain Natriuretric Peptide (BNP) was found, the value of plasma BNP concentration as the indicator in diagnosis and prognosis of heart failure has been confirmed. The importance of BNP measurement was emphasized in the guideline of heart failure issued by ESC and ACC/AHA. Now, BNP becomes the potential biomarker in diagnosis, risk stratification and prognosis evaluation of heart failure.Aims: To obtain the hybridoma secreting BNP monoclonal antibodies, and to prepare monoclonal antibodies against BNP.Methods: The synthesized BNP was conjugated to bovine serum albumin (BSA) by EDC coupling procedure to form complete antigen (BNP-BSA). The Balb/c mice were first immunized by intraperitoneal injection with BNP-BSA (100 μg BNP), which was emulsified in an equal volume of complete Freund's adjuvant. Three weeks later, the mice were boosted with the same amount of BNP-BSA emulsified with incomplete Freund's adjuvant at three weeks interval. The titer of the BNP antibodies in the immunized mice sera was determined by indirect enzyme-linked immunosorbent assay (ELISA). The mice ware boosted again with BNP-BSA without adjuvant three days before fusion.About 1×10~8 splenic lymphocytes from the immunized mouse were fused with 2-5×10~7 myeloma SP2/0 cells in the presence of polyethylene glycol. The mixed cells were plated in 96-well plates and grown in HAT (hypoxanthine,...