Synthesis of a Neu2en5Ac analog hapten and isolation of monoclonal antibody to Neu2en5Ac.
Neu2en5Ac is a minor component of body fluids and is abundant in sialuria, but no antibody to detect it has been reported. 5-Acetamido-2,6-anhydro-9-glutaramido-3,5,9-trideoxy-D-glycero-D- galacto-non-2-enonic acid has been synthesized and conjugated with keyhole limpet hemocyanin (KLH) for immunization. A hybridoma named SIC172 was obtained that produces a monoclonal antibody (MAb) to Neu2en5Ac. SIC172 MAb in culture supernatant bound strongly to the hapten conjugated to BSA in ELISA, but slightly to fetuin, a glycoprotein which is rich in Neu5Ac. SIC172 MAb (IgG3(kappa)), purified with a protein A/G affinity column, bound strongly to fetuin. Neu2en5Ac competed with the MAb in binding in amounts as low as 3 microM, while the competition of Neu5Ac appeared at amounts of more than 300 microM. SIC172 MAb is a unique MAb specific to Neu2en5Ac and might be useful for detecting Neu2en5Ac, which occurs naturally and in sialuria.
Laboratory of Life Science & Biomolecular Engineering, Japan Tobacco Inc., Yokohama, Japan. hiroya.kamei@ims.jti.co.jp
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Kamei
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Kajihara
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Nishi
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D002240:Carbohydrate Sequence; D004305:Dose-Response Relationship, Drug; D004306:Dose-Response Relationship, Immunologic; D006241:Haptens / Q000276:immunology*; D006801:Humans; D006825:Hybridomas; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000276:immunology; D013154:Spleen / Q000378:metabolism
D000911:Antibodies, Monoclonal; D006241:Haptens; C036137:2-deoxy-2,3-dehydro-N-acetylneuraminic acid; D019158:N-Acetylneuraminic Acid