Production and characterization of monoclonal antibodies against a novel 1α,25-dihydroxyvitamin D 3-bovine serum albumin conjugate linked through the 11α-position

Abstract

To obtain an antibody which is useful for developing a selective immunoaffinity chromatography (IAC) of 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3], which is an effective pretreatment tool for determining this metabolite in biological fluids, a variety of monoclonal antibodies have been produced against a novel haptenic derivative, 11α-hemiglutaryloxy-1α,25-dihydroxyvitamin D 3, linked to bovine serum albumin. BALB c , A J and C57BL 6 mice, as well as Wistar and SD rats, were immunized with the hapten-carrier conjugate and the titer of the anti-1,25(OH) 2D 3 antibody in serum was examined. Spleen cells from A J mice or SD rats, which showed a high titer, were fused with P3/NS1/1-Ag4-1 myeloma cells. After serial screening by enzyme-linked immunosorbent assay and radioimmunoassay, sixteen kinds of hybridoma clones secreting different anti-1,25(OH) 2D 3 antibodies were established. Scatchard analysis revealed that 12 antibodies out of 16 have enough affinity to 1,25(OH) 2D 3 ( K a>10 9 M −1). By a cross-reaction study using various related compounds, two antibodies, M114 and M129, both derived from A J mice, were found to have suitable specificity. Although they showed relatively high cross-reactivities with the 1,25(OH) 2D 3 derivatives having 24-hydroxylated (both 20%) or lactonized (30%, 8.9%) side chain, all the tested 1α-deoxy-type metabolites, some of which interfere with the 1,25(OH) 2D 3 assays, were successfully discriminated [25(OH)D 3: 0.67%, 0.40%; 24,25(OH) 2D 3: 0.27%, 0.24%; 25(OH)D 3 3-sulfate: 0.11%, 0.14%]. These monoclonal antibodies will be applicable for developing not only IAC but also various immunoassay procedures.