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

BACKGROUND: 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). METHODS/PRINCIPLE FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.