Characterization of the antigenic specificity of four different anti-(α2→8-linked polysialic acid) antibodies using lipid-conjugated oligo/polysialic acids

Abstract

A rapid, sensitive, and facile method for screening and characterizing anti-polysialic acid (polySia) antibodies using lipid-conjugated oligo/polysialic acids (oligo/poly-Sia) was developed, which is based on an enzyme-linked immunosorbent assay. Homooligo/polymers of α2→8-linked N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid, and 2-keto-3-deoxy-D-galacto-nononic acid (KDN) were conjugated with phosphatidylethanolamine dipalmitoyl (PE) by reductive amination to prepare neo-oligo/polysialoglycolipids (oligo/polySia-PE). Using this method, the anti-polySia equine antibody, H.46, bound to (→8Neu5Acα2→)n-PE, where n = 9 or more residues, a result in confirmation of previous binding studies using radiolabeled oligo/polyNeu5Ac. The antigenic specificity and sensitivity of two monoclonal anti-poly/oligoNeu5Ac antibodies (mAb.12E3 and mAb.5A5) and one anti-oligoKDN antibody (mAb.kdn8kdn), were also determined. mAb.12E3 could detect as little as 25 pg/well of oligo/polyNeu5Ac-PE, while mAb.5A5 and polyclonal antibody H.46 required at least 0.4 ng/well of oligo/polyNeu5Ac-PE to be detected. mAb.kdn8kdn detected as little as 12 ng/well of oligoKDN-PE. Using a series of oligo/polySia-PE with defined degrees of polymerization (DP), the minimum chain length for immunoreactivity of the anti-polySia antibodies was determined to be: DP 5 for mAb.12E3; DP 3 for mAb.5A5; DP 2 for mAb.kdn8kdn; and DP 8 for H.46. Thus, mAb.12E3 and mAb.5A5 recognize shorter oligomers of Neu5Ac than H.46, a finding that is of practical value for identifying shorter oligoSia chains in glycoconjugates. Because mAb.12E3 and mAb.5A5 also recognize extended polySia chains, these antibodies cannot be used, however, to differentiate between short and long chains of polySia when both are expressed on the same molecule.