Figure 4.

Cloned antibodies specificity against multiple glycan antigens. (a) Structures of SLe^a and closely-related glycans. In AcSLe^a (A), the N-acetyl group on GlcNAc (shaded grey circle) is oriented to the same side as the sialic acid (purple diamond), while in opposite directions in the AcSLe^x (B) isomer. (b) Specificity of native and RA9-mutated clones expressed as full-length IgG were examined by ELISA. 96-well plate was coated with PAA-polymer conjugated glycans, and primary antibodies examined at 10 ng/μL. Relative optical density (OD) was calculated as percentage of maximal binding of each antibody clone, followed by averaging the relative rank of two independent experiments (mean ± SEM). Glycans labeled A–F correspond to the structures described in (a), G is polyacrylamide backbone (PAA) control. (c) Binding of native and RA9-23 IgGs against diverse glycans was examined at 0.16, 0.08 and 0.032 ng/μL by a sialoglycan microarray (List of glycans in Table S1). Relative fluorescence units (RFU) was calculated as percentage of maximal binding at each concentration, followed by averaging the relative RFU rank of the three tested antibody concentrations for each glycan (mean ± SEM). A–C, E, H–J correspond to the structures described in (a). (d) Chemical structures of sialic acid types Neu5Ac (Ac), Neu5Gc (Gc), 9-O-acetyl-Neu5Ac (9-O-Ac), and 9-O-acetyl-Neu5Gc (9-O-Gc). (e) Specificity of the full-length antibody mutant clone RA9-23 was examined by ELISA inhibition assay against coated SLe^a-PAA-Biotin, after pre-incubation of the antibody with specific (SLe^a) or non-specific glycans (SLe^x and Le^a). **** p < 0.001.