# Conjugates of α-d-Galp-(1→3)-β-d-Galp for the serological diagnosis of Chagas disease

## TL;DR

Researchers developed a new diagnostic tool for Chagas disease using a disaccharide biomarker that shows high sensitivity and accuracy.

## Contribution

A novel chemical synthesis and conjugation protocol for a T. cruzi glycotope with potential for improved Chagas disease diagnosis.

## Key findings

- The disaccharide α-D-Galp-(1→3)-β-D-Galp conjugated to BSA showed high sensitivity and accuracy (AUC = 0.905) in distinguishing ChD patient sera from healthy controls.
- Conjugation to T. cruzi antigenic peptides confirmed the biomarker's utility and enabled bivalent diagnostic reagents.
- The disaccharide is a robust biomarker for T. cruzi infection with minor cross-reactivity concerns.

## Abstract

Chagas disease (ChD), caused by the parasitic protozoan Trypanosoma cruzi, is a lifelong, neglected tropical disease with substantial medical and socioeconomic impact. Despite this situation, currently available diagnostic and therapeutic methods display serious limitations. A promising strategy to improve ChD serodiagnosis involves targeting parasite carbohydrate antigens, particularly the α-galactosyl–rich mucins that coat the surface of bloodstream trypomastigotes (tGPI-mucins).

Here, we present a concise and efficient protocol for the chemical synthesis of a tGPI-mucin–derived glycotope, the disaccharide α-D-Galp-(1 → 3)-β-D-Galp, and its functional conjugation to different scaffolds using the squarate method. A neoglycoprotein made upon a bovine serum albumin (BSA) carrier decorated with ∼38 units of the disaccharide, termed BSA-Di, was interrogated with sera of chronic ChD patients and healthy individuals from Argentina using an in-house enzyme-linked immunosorbent assay (ELISA). BSA-Di exhibited excellent sensitivity and effectively discriminated between ChD-positive and negative sera with high accuracy (AUC = 0.905), though its specificity was partially affected by cross-reactivity of some non-ChD sera containing natural α-Gal antibodies. Conjugation of α-D-Galp-(1 → 3)-β-D-Galp to T. cruzi antigenic peptides, instead of BSA, corroborated these findings and enabled the generation of bivalent ChD diagnostic reagents combining glycan- and peptide-based epitopes.

Overall, our results identify α-D-Galp-(1 → 3)-β-D-Galp as a robust and reliable biomarker of T. cruzi infection. The methodologies and tools described here, together with optimized derivatives, are expected to positively impact ChD serological applications.

Despite the enormous burden imposed by Chagas disease, diagnostic and therapeutic methods still present serious deficiencies. Towards filling this gap, we herein developed a protocol for the chemical synthesis of α-D-Galp-(1 → 3)-β-D-Galp, a major glycotope present on the Trypanosoma cruzi surface coat. This disaccharide was conjugated with different molecular scaffolds and serologically evaluated using an in-house enzyme-linked immunosorbent assay (ELISA). Our results indicate that α-D-Galp-(1 → 3)-β-D-Galp provides an overall robust and reliable biomarker of T. cruzi infection, with excellent sensitivity and only minor concerns regarding its potential cross-reactivity with ‘natural’ α-Gal antibodies. These findings indicate that the tools developed here, as well as optimized versions derived from them, should have a positive impact on the diagnosis and clinical management of Chagas disease and on the identification and/or clinical validation of novel drug/vaccine candidates for the treatment of T. cruzi infections.