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. Author manuscript; available in PMC: 2019 Jun 24.
Published in final edited form as: Methods Mol Biol. 2019;1955:287–308. doi: 10.1007/978-1-4939-9148-8_22

Fig. 3.

Fig. 3

Synthesis of the α-Gal-containing NGP11b, and comparative immunoreactivity analysis between NGP11b and tGPI-mucins. (a) Synthesis of the trisaccharide 3-thiopropyl α-d-galactosyl-(1→2)-[α-d-galactosyl-(1→6)]-β-d-galactoside and its conjugation to maleimide-derivatized BSA to afford the NGP11b. (b) MALDI-TOF-MS spectra of BSA and NGP11b. Singly ([BSA]1+ and [NGP11b]1+) and doubly ([BSA]2+ and [NGP11b]2+) charged molecular ions of BSA and NGP11b are indicated. m/z, mass to charge ratio. (c) CL-ELISA immunoreactivity of ChSP, NHSP, Ch anti-α-Gal Abs, and NHS anti-α-Gal Abs to immobilized NGP11b. A dose-dependent reactivity was observed with ChSP, NHSP, and Ch anti-α-Gal Abs. A clear differential immunoreactivity was observed between these sera and Abs. (d) Reactivity of sera from three individual chronic ChD patients to tGPI-mucins and NGP11b, before and after α-galactosidase (α-Galase) treatment. The considerable reduction in reactivity following enzymatic treatment indicates that specificity of individual Ch sera is directed mainly to the terminal, nonreducing α-Gal residues or α-Gal-containing glycotopes present in both tGPI-mucins and NGP11b. (e) Follow-up of patients before (−) and 24 months after (+) benznidazole (BZN) treatment, using tGPI-mucins or NGP11b as Ags in CL-ELISA. ChSP (c) and individual ChS samples (Patients # 45, 60, and 221) (d) were from IMT/FM/UCV. NHSP (c) and individual ChS samples (Patients # 4, 59, and 60) (e) were from ISGlobal-Barcelona. Serum samples were collected strictly following the International Ethical Guidelines for Biomedical Research Involving Human Subjects and protocols approved by the Institutional Review Boards of IMT/FM/UCV and ISGlobal-Barcelona.