Seferian Karina R, Tamm Natalia N, Semenov Alexander G, Tolstaya Anastasia A, Koshkina Ekaterina V, Krasnoselsky Mihail I, Postnikov Alexander B, Serebryanaya Daria V, Apple Fred S, Murakami MaryAnn M, Katrukha Alexey G
HyTest Ltd., Turku, Finland.
Clin Chem. 2008 May;54(5):866-73. doi: 10.1373/clinchem.2007.100040. Epub 2008 Mar 13.
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.
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.
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.
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.
脑钠肽（BNP）或NT-proBNP（BNP前体的N端片段）检测被推荐用于辅助心力衰竭患者的诊断和预后评估。最近有研究表明，proBNP在人体血液中发生了O-糖基化修饰。本研究的目的是确定NT-proBNP分子上那些在最佳NT-proBNP检测中应被抗体识别的位点。
我们使用了一组针对NT-proBNP分子不同表位的单克隆抗体，通过多种免疫化学方法分析内源性NT-proBNP。
用糖苷酶混合物处理内源性NT-proBNP后，去糖基化的NT-proBNP与针对该分子中段的单克隆抗体（MAbs）之间的相互作用显著改善。针对NT-proBNP N端和C端部分（表位13 - 24和63 - 76）的MAbs能够以相似的效率识别糖基化和去糖基化的蛋白。
内源性NT-proBNP的中央部分发生了糖基化修饰，这使得它对于针对该分子中段的抗体几乎“不可见”。因此，即使使用一种针对该分子中央部分的抗体（多克隆或单克隆）进行夹心检测，也可能低估内源性NT-proBNP的实际浓度。针对NT-proBNP N端和C端部分（表位13 - 24和63 - 76）的MAbs是用于最佳NT-proBNP免疫检测的最佳候选抗体。
