Total NT-proBNP assay, a new immunoturbidimetric assay utilizying antibodies targeting glycosylation-free regions of NT-proBNP

Introduction

Commercial NT-proBNP immunoassays often use antibodies targeting the central region of NT-proBNP, which is frequently glycosylated. Glycosylation affects antibody binding and may cause substantial underestimation of NT-proBNP, as up to 80% of NT-proBNP can be glycosylated. The aim of this study was to evaluate and confirm that Gentian NT-proBNP assay is unaffected by glycosylation and compare it with comercially available assays.

Methods

To assess total NT-proBNP, independent of glycosylation, plasma samples were analysed either untreated or after treatment with a mixture of glycosidases (24 h, 37°C). Anonymized patient samples were measured using a commercial assay targeting glycosylated region and the Gentian prototype targeting glycosylation-free regions of NT-proBNP. In addition, NT-proBNP levels were assessed in 60 healthy individuals (30 women, 30 men), stratified into three age groups (<45, 45–65,5 years; n=10 per gender per group).

Results

NT-proBNP concentrations measured in treated and untreated plasma samples showed no significant effect of glycosidase treatment when analysed with the Gentian assay prototype. Values in treated and untreated samples were highly correlated (slope = 0.849, R² = 0.936). In contrast, the commercial assay markedly underestimated NT-proBNP in untreated samples, with a slope of 6.34. Results from glycosidase-treated samples measured by the commercial assay correlated more closely with the Gentian assay (R² = 0.889) than non-treated samples, confirming the impact of glycosylation on NT-proBNP detection.

In healthy individuals, NT-proBNP levels measured with the Gentian assay were significantly higher than values obtained with commercial assays. NT-proBNP concentrations increased with age, with median values rising from 1407 ng/L (IQR 945–1713 ng/L) in individuals <45 years, to 1878 ng/L (IQR 1545–2192 ng/L) in those aged 45–65 years, and 2332 ng/L (IQR 1509–2725 ng/L) in individuals >65 years. NT-proBNP concentrations differed significantly across age groups (p = 0.0009), demonstrating an overall increase in NT-proBNP levels with age.

NT-proBNP concentrations were slightly higher in women. For women (n = 30), the median value was 1807 ng/L with an IQR of 1484–2281 ng/L. For men (n = 30), the median was 1633 ng/L (IQR 1115–2281 ng/L). However, the gender related variation was not statistically significant (p = 0.175). In the age-stratified analyses, women exhibited consistently higher NT-proBNP concentrations across all age groups (<45, 45–65, >65), but none of the differences reached statistical significance (<45 years, p=0.31; 45–65 years, p=0.72; >65 years: p=0.26).

Conclusions

These findings confirm that glycosylation leads to systematic underestimation of NT-proBNP by assays targeting glycosylated regions. Glycosylation-independent immunoassays detect endogenous NT-proBNP regardless of glycosylation status and may offer clinical advantages for heart failure diagnosis and management.