A-010 Development and diagnostic utility of immunoturbidimetric NT-proBNP assay based on antibodies targeting glycosylation-free regions of NT-proBNP
Authors: Havelka A , Tran L T , Schmelck V , Di Domenica M , Nilsen T , Knüttel T 
Published: October 2024

Abstract

Background
Multiple commercial immunoassays are available for the measurement of circulating NT-proBNP levels. Most of the assays use antibodies that bind to the central region of NT-proBNP, which is usually glycosylated. Glycosylation of NT-proBNP restricts the binding of the antibodies and detection of the protein. It has been shown that up to 80% of the circulating NT-proBNP may be glycosylated in the central region. Several studies have confirmed that NT-proBNP assays underestimate the concentration of plasma NT-proBNP measuring only a subfraction of the circulating NT-proBNP because of the negative effect of glycosylation on NT-proBNP recognition by the antibodies. Aim of this study is to develop an NT-proBNP assay which is not affected by glycosylation of the protein and comparison of its diagnostic utility with performance of other commercially available immunoassays.

Methods
To determine the total concentration of NT-proBNP, independent of glycosylation, plasma samples were either non-treated or treated with a mixture of glycosidases for 16 h. Following, samples were analyzed by NT-proBNP immunoassay based on antibodies targeting a glycosylated region of NT-proBNP (Siemens Healthineers, Immulite NT-proBNP) and the NT-proBNP turbidimetric assay based on antibodies targeting regions of NT-proBNP that are free of O-glycans (in development by Gentian).

Results
As Gentian NT-proBNP assay is based on antibodies targeting non-glycosylated regions of NT-proBNP, we expected to see no or little effect of deglycosylation on NT-proBNP concentration. NT-proBNP was measured in 57 plasma samples either treated or untreated with a mixture of glycosidases. No significant effect of the glycosidase treatment was detected on NT-proBNP values measured by Gentian developed NT-proBNP assay. NT-proBNP concentrations in treated and non-treated samples were similar (slope of 0.849), highly correlated (R2=0.926) and showed an average ratio of NT-proBNP concentrations between deglycosylated vs. non-treated of 1.6 +/- 1.3. As a comparison, we used one of the commercially available assays with antibodies targeting the central region of the NT-proBNP, Immulite NT-proBNP (Siemens Healthineers). NT-proBNP concentrations showed a good correlation (R2=0.879), but the assay strongly underestimated concentrations in untreated samples (slope of 2.555) with an average ratio of deglycosylated vs. non-treated of 3.4 +/- 1.8.

Conclusions
The measurement of NT-proBNP has become a widely adopted method for diagnosing heart failure (HF). Given the considerable variability in levels and site occupancy of O-glycosylated proteins, it is reasonable to anticipate that immunoassays targeting glycosylation-free regions of NT-proBNP may offer advantages in HF diagnosis, prognosis, and therapy monitoring for specific patient groups and disease states since these assays can detect endogenous NT-proBNP regardless of its glycosylation status. Further investigations, including exploration of the clinical significance are necessary in order to understand potential benefits and applications of such assays in management of patients with HF.