CHARACTERIZATION OF THE ANTIGENIC SPECIFICITY OF 4 DIFFERENT ANTI-(ALPHA-2-]8-LINKED POLYSIALIC ACID) ANTIBODIES USING LIPID-CONJUGATED OLIGO POLYSIALIC ACIDS

A rapid, sensitive, and facile method for screening and characterizing anti-polysialic acid (polySia) antibodies using lipid-conjugated olig o/polysialic acids (oligo/polySia) was developed, which is based on an enzyme linked immunosorbent assay, Homooligo/polymers of alpha 2-->8- linked N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid, an d 2-keto-3-deoxy-D-galacto-nononic acid (KDN) were conjugated with pho sphatidylethanolamine dipalmitoyl (PE) by reductive amination to prepa re neo-oligo/polysialoglycolipids (oligo/polySia-PE), Using this metho d, the anti-polySia equine antibody, H.46, bound to (-->8Neu5Ac alpha 2-->)(n)-PE, where n = 9 or more residues, a result in confirmation of previous binding studies using radiolabeled oligo/polyNeu5Ac. The ant igenic specificity and sensitivity of two monoclonal anti-poly/oligoNe u5Ac antibodies (mAb.12E3 and mAb.5A5) and one anti-oligoKDN antibody (mAb.kdn8kdn), were also determined, mAb.12E3 could detect as little a s 25 pg/well of oligo/polyNeu5Ac-PE, while mAb.5A5 and polyclonal anti body H.46 required at least 0.4 ng/well of oligo/polyNeu5Ac-PE to be d etected, mAb.kdn8kdn detected as little as 12 ng/well of oligoKDN-PE, Using a series of oligo/polySia-PE with defined degrees of polymerizat ion (DP), the minimum chain length for immunoreactivity of the anti-po lySia 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 glycoco njugates, Because mAb.12E3 and mAb.5A5 also recognize extended polySia chains, these antibodies cannot be used, however, to differentiate be tween short and long chains of polySia when both are expressed on the same molecule.