Determination of the specificity of monoclonal antibodies against Schistosoma mansoni CAA glycoprotein antigen using neoglycoconjugate variants.

The immunogenic O-glycan of circulating anodic antigen (CAA) is a high-molecular-mass polysaccharide with the unique →6)-[β-D-Glc pA-(1→3)]-β-D-Gal pNAc-(1→ repeating unit. To obtain information at the molecular level about the specificity of monoclonal antibodies against CAA, the immunoreactivity of two series of bovine serum albumin-coupled synthetic oligosaccharides related to the CAA O-glycan was monitored using ELISA and surface plasmon resonance spectroscopy. The importance of the axial hydroxyl group of β-D-Gal pNAc for antibody binding was investigated using the following series of analogues: β-D-Glc pA-(1→3)-β-D-Glc pNAc-(1→O); β-D-Glc pNAc-(1→6)-[β-D-Glc pA-(1→3)]-β-D-Glc pNAc-(1→O); and β-D-Glc pA-(1→3)-β-D-Glc pNAc-(1→6)-[β-D-Glc pA-(1→3)]-β-D-Glc pNAc-(1→O). In the second series of analogues, β-D-Glc p6 S-(1→3)-β-D-Gal pNAc-(1→O), β-D-Gal pNAc-(1→6)-[β-D-Glc p6 S-(1→3)]-β-D-Gal pNAc-(1→O), and β-D-Glc p6 S-(1→3)-β-D-Gal- pNAc-(1→6)-[β-D-Glc p6 S-(1→3)]-β-D-Gal pNAc-(1→O), the native β-D-Glc pA moiety was replaced by β-D-Glc p6 S to evaluate the influence of the nature of the charge on antibody recognition. Comparison of the immunoreactivity of these series with that measured for conjugates containing corresponding synthetic CAA fragments showed that the antibody binding levels can be correlated to the antibody specificity to CAA fragments. For the most reactive antibodies, the structural changes chosen (β-D-Gal pNAc replaced by β-D-Glc pNAc, and β-D-Glc pA replaced by β-D-Glc p6 S) completely eradicated the binding. [ABSTRACT FROM AUTHOR]