Immunodetection of glycosylated NT-proBNP circulating in human blood.

Seferian KR; Tamm NN; Semenov AG; Tolstaya AA; Koshkina EV; Krasnoselsky MI; Postnikov AB; Serebryanaya DV; Apple FS; Murakami MM; Katrukha AG

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Article

Title:	Immunodetection of glycosylated NT-proBNP circulating in human blood.
Authors:	Seferian KR; Tamm NN; Semenov AG; Tolstaya AA; Koshkina EV; Krasnoselsky MI; Postnikov AB; Serebryanaya DV; Apple FS; Murakami MM; Katrukha AG
Publication:	Clin Chem. 2008 May;54(5):866-73. doi: 10.1373/clinchem.2007.100040. Epub 2008 Mar 13.
PubmedID	18339697
Abstract
BACKGROUND: Brain natriuretic peptide (BNP) or NT-proBNP (N-terminal fragment of BNP precursor) measurements are recommended as aids in diagnosis and prognosis of patients with heart failure. Recently it has been shown that proBNP is O-glycosylated in human blood. The goal of this study was to map sites on the NT-proBNP molecule that should be recognized by antibodies used in optimal NT-proBNP assays. METHODS: We analyzed endogenous NT-proBNP by several immunochemical methods using a broad panel of monoclonal antibodies specific to different epitopes of the NT-proBNP molecule. RESULTS: Treatment of endogenous NT-proBNP by a mixture of glycosidases resulted in significant improvement of the interaction between deglycosylated NT-proBNP and monoclonal antibodies (MAbs) specific to the mid-fragment of the molecule. MAbs specific to the N- and C-terminal parts of NT-proBNP (epitopes 13-24 and 63-76) were able to recognize glycosylated and deglycosylated protein with similar efficiency. CONCLUSIONS: The central part of endogenous NT-proBNP is glycosylated, making it almost "invisible" for the antibodies specific to the mid-fragment of the molecule. Thus sandwich assays using even one antibody (poly- or monoclonal) specific to the central part of the molecule could underestimate the real concentration of endogenous NT-proBNP. MAbs specific to the N- and C-terminal parts of NT-proBNP (epitopes 13-24 and 63-76) are the best candidates to be used in an assay for optimal NT-proBNP immunodetection.