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(κ)), 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 μM, while the competition of Neu5Ac appeared at amounts of more than 300 μM. SIC172 MAb is a unique MAb specific to Neu2en5Ac and might be useful for detecting Neu2en5Ac, which occurs naturally and in sialuria.