Assay for measurement of intact B-type natriuretic peptide prohormone in blood

B-Type natriuretic peptide (BNP1-32) as well as the N-terminal fragment of the prohormone containing residues 1-76 (NT-proBNP1-76), both cleavage products of the precursor proBNP1-108, are reported to be powerful markers for prognosis and risk stratification of heart failure. However, the intact precursor also circulates in the bloodstream. Assays for the detection of these cleavage products have been developed, but most of these assays may overestimate the concentrations of the cleavage products because they also measure the precursor form. It is therefore important to develop an immunoassay that specifically measures solely proBNP1-108 in plasma.

After carefully designing the peptide used to immunize mice, we selected a specific monoclonal antibody (mAb Hinge76) that recognizes the cleavage site of proBNP1-108, an epitope present only in the precursor form. mAb Hinge76 recognizes recombinant proBNP1-108 in a dose-dependent manner, without any significant cross-reactivity with either recombinant NT-proBNP1-76 or synthetic BNP1-32. By combining mAb Hinge76 with a polyclonal antibody directed against BNP1-32, we were able to set up a proBNP1-108-specific sandwich immunoassay able to confirm the presence of proBNP1-108 in blood samples.

From a cohort of 50 healthy persons and 170 patients with congestive heart failure (CHF), our assay was able to differentiate healthy individuals from CHF patients (P <0.005). Interestingly, plasma proBNP1-108 concentrations were correlated with New York Heart Association classification. Moreover, a close relationship between proBNP1-108 and BNP1-32 concentrations may exist, as a good correlation (r2= 0.89) was obtained when their respective concentrations were compared.

mAb Hinge76 is the first proBNP1-108-specific mAb produced that allows accurate estimation of proBNP1-108 concentrations in plasma.