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Clinical Chemistry 54: 866-873, 2008. First published March 13, 2008; 10.1373/clinchem.2007.100040
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(Clinical Chemistry. 2008;54:866-873.)
© 2008 American Association for Clinical Chemistry, Inc.

Proteomics and Protein Markers

Immunodetection of Glycosylated NT-proBNP Circulating in Human Blood

Karina R. Seferian1,a, Natalia N. Tamm1, Alexander G. Semenov2, Anastasia A. Tolstaya3, Ekaterina V. Koshkina4, Mihail I. Krasnoselsky5, Alexander B. Postnikov1, Daria V. Serebryanaya1, Fred S. Apple6, MaryAnn M. Murakami6 and Alexey G. Katrukha1

1 HyTest Ltd., Turku, Finland; 2 Department of Biochemistry, Moscow State University, Moscow, Russia; 3 Moscow Research Institute of Medical Ecology, Moscow, Russia; 4 67 City Hospital, Moscow, Russia; 5 Moscow State Medico- Stomatological University, Moscow, Russia; 6 Hennepin County Medical Center, University of Minnesota School of Medicine, Department of Laboratory Medicine and Pathology, Minneapolis, MN.

aAddress correspondence to this author at: HyTest Ltd., Intelligate, 6th floor, Joukahaisenkatu 6, 20520 Turku, Finland. Fax +358 25120909; e-mail karina.seferian{at}hytest.fi.

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.






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