Samman Butt Inayat Ali Khan Mehreen Umair Tauseef Ali Memon Ehsan Ulhaq Muhammad Ahmed Butt


Background: Heart failure (HF) diagnosis often relies on peptides originating from the BNP precursor, including BNP, proBNP, and the NT-proBNP, serving as key markers. However, there remains ambiguity regarding the specific forms of these peptides present in the bloodstream and their detectability through existing assays. Objective: To increase our understanding and knowledge of the circulating forms of BNP peptides in HF and their implications for diagnostic and prognostic assessment. Study design: An analytical cross-sectional study. Place and Duration: This study was conducted in The Indus Hospital and Health Network Karachi from March 2023 to March 2024. Methodology: In this research, we developed innovative assays for detecting NT-proBNP, proBNP, and BNP using specific monoclonal antibodies designed for their recognition. These antibodies were thoroughly tested in dual-site-combinations by using time-resolved immunoassays. We employed synthetic antigens and recombinant antigens, as well as plasma samples from HF patients. Furthermore, we utilized gel filtration fast protein liquid chromatography (FPLC) to analyse proBNP and related molecules in both pre- and post-protein fractionated HF plasma extracts and samples, using Sep-Pak C18 cartridges. Results: Our investigations demonstrated specific detection limits for proBNP, BNP, and NT-proBNP assays, measured at 0.4, 3, and 10 ng/L, respectively. Following analyses using gel filtration-FPLC, distinct peaks emerged, with one each for NT-proBNP (25 kDa) and proBNP (37 kDa), and two for BNP immunoreactivity. Particularly noteworthy was the finding that in patient plasma, the molar concentration of NT-proBNP exceeded that of proBNP by almost tenfold. The mean proBNP:BNP ratio in patient plasma was calculated at 6.3, with variations ranging from 1.8 to 10.8. Conclusions: Our findings highlight proBNP as the principal BNP-immunoreactive form circulating in human blood. Moreover, we underscore the influence of sample handling techniques and assay methodologies on determining the proBNP:BNP ratio in plasma samples, emphasizing the importance of standardized protocols in peptide measurement.

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Heart Failure Biomarkers, Probnp Detection Assays, NT-Probnp Quantification, Peptide Immunoreactivity, Plasma Sample Analysis

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