CHO-131 recognition of the glycan structure C2-O-sLe^X.

(A) Structures of glycopeptides used in the enzyme-linked immunosorbent assay (ELISA). Glycopeptides corresponding to the N-terminal peptide sequence of human PSGL-1 and the O-glycan structures C2-O-sLe^X (GP-6), C1-O-sLe^X (GP-6′), C2-O-sLN (GP-5), or GalNAc (GP-1) at threonine residue 57 (arrow) were generated by synthetic methods. (B) Glycopeptide ELISA. Microtiter wells were coated with individual glycopeptides as indicated, blocked with BSA, and incubated with the mAbs CHO-131, HECA-452 (anti-sLe^X), or PL1 (anti–PSGL-1 peptide). Bound antibodies were detected by incubation with horseradish peroxidase–conjugated, goat anti–mouse IgM (CHO-131) or IgG (PL1), or goat anti–rat IgM (HECA-452) followed by incubation with ABTS/peroxidase substrate and absorbance measurements at 405 nm. (C) CHO-131 specificity at different antibody concentrations. ELISA assay was performed as in panel B with the use of different concentrations of CHO-131 (0, 0.05, 0.5, 5, 10 μg/mL).