Direct Immunochemiluminescent Assay for proBNP and Total BNP in Human Plasma proBNP and Total BNP Levels in Normal and Heart Failure

BACKGROUND: Recent studies have shown that in addition to brain (or B-type) natriuretic peptide (BNP) and the N-terminal proBNP fragment, levels of intact proBNP are also increased in heart failure. Moreover, present BNP immunoassays also measure proBNP, as the anti-BNP antibody cross-reacts with proBNP. It is important to know the exact levels of proBNP in heart failure, because elevation of the low-activity proBNP may be associated with the development of heart failure. METHODOLOGY/PRINCIPAL FINDINGS: We therefore established a two-step immunochemiluminescent assay for total BNP (BNP+proBNP) and proBNP using monoclonal antibodies and glycosylated proBNP as a standard. The assay enables measurement of plasma total BNP and proBNP within only 7 h, without prior extraction of the plasma. The detection limit was 0.4 pmol/L for a 50-µl plasma sample. Within-run CVs ranged from 5.2%-8.0% in proBNP assay and from 7.0%-8.4% in total BNP assay, and between-run CVs ranged from 5.3-7.4% in proBNP assay and from 2.9%-9.5% in total BNP assay, respectively. The dilution curves for plasma samples showed good linearity (correlation coefficients = 0.998-1.00), and analytical recovery was 90-101%. The mean total BNP and proBNP in plasma from 116 healthy subjects were 1.4 ± 1.2 pM and 1.0 ± 0.7 pM, respectively, and were 80 ± 129 pM and 42 ± 70 pM in 32 heart failure patients. Plasma proBNP levels significantly correlate with age in normal subjects. CONCLUSIONS/SIGNIFICANCE: Our immunochemiluminescent assay is sufficiently rapid and precise for routine determination of total BNP and proBNP in human plasma.