A sandwich ELISA for assessment of pharmacokinetics of HSA-(BNP)2 fusion protein in mouse plasma

Brain natriuretic peptide (BNP) is a circulating hormone of cardiac origin that plays an important role in the regulation of intravascular blood volume and vascular tone. HSA-(BNP)2, derived from the joining of human BNP to the C-terminus of human serum albumin (HSA), has been developed to prolong the BNP pharmacodynamic action. For the analysis of pharmacokinetics of the new drug, a novel sandwich enzyme-linked immunosorbent assay (ELISA) was established and validated to quantify HSA-(BNP)2 fusion protein in mouse plasma. The ELISA method was calibrated with 1:10 and 1:100 dilutions of blank mouse plasma spiked with HSA-(BNP)2 standard and validated with respect to parallelism, precision (intra- and inter-assay variation), accuracy (recovery), specificity and stability. The practical working range was estimated to be 31.2–2000 ng/ml with the limit of detection was 7.8 ng/ml. Recoveries ranged from 80.5 to 108.4%, while the intra- and inter-assay precisions were <2.73% and <4.32%, respectively. The terminal half-life of HSA-(BNP)2 was 2.14 h, which had extended more than 40 times compared to 3.1 min half-life of BNP monomer in mouse.