Immunodetection of glycosylated NT-proBNP circulating in human blood.
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.
Seferian
Karina R
KR
HyTest Ltd., Turku, Finland.
Tamm
Natalia N
NN
Semenov
Alexander G
AG
Tolstaya
Anastasia A
AA
Koshkina
Ekaterina V
EV
Krasnoselsky
Mihail I
MI
Postnikov
Alexander B
AB
Serebryanaya
Daria V
DV
Apple
Fred S
FS
Murakami
MaryAnn M
MM
Katrukha
Alexey G
AG
D000911:Antibodies, Monoclonal; D002850:Chromatography, Gel; D005455:Fluorescent Antibody Technique / Q000379:methods; D006031:Glycosylation; D006333:Heart Failure / Q000097:blood; D006801:Humans; D020097:Natriuretic Peptide, Brain / Q000097:blood* / Q000276:immunology; D010446:Peptide Fragments / Q000097:blood* / Q000276:immunology
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; C109794:pro-brain natriuretic peptide (1-76); D020097:Natriuretic Peptide, Brain