Production and Characterization of Monoclonal Antibodies against Two Haptenic Derivatives of 1α, 25-Dihydroxyvitamin D₃ Conjugated with Bovine Serum Albumin through the C-3 or C-24 Position

Abstract

To develop the immunochemical methods for determining 1α, 25-dihydroxyvitamin D₃ [1, 25(OH)₂D₃] in clinical samples, a variety of monoclonal anti-1, 25(OH)₂D₃ antibodies have been generated. Two kinds of hapten-carrier conjugates, 25-hydroxyvitamin D₃ 3-hemisuccinate (hapten 3-HS) and 1α-hydroxy-25, 26, 27-trinorvitamin D₃ 24-oic acid (hapten 24-OA) conjugated with bovine serum albumin, were used for immunization. Spleen cells from SD rats or BALB/c mice, each immunized with the conjugate of hapten 3-HS or 24-OA, were fused with P3/NS1/1-Ag4-1 myeloma cells. After screening by ELISA employing β-galactosidase-labeled haptens, seven kinds of hybridomas secreting anti-1, 25(OH)₂D₃ antibodies were established. Binding characteristics of these antibodies (K_a 0.73-20×10⁹ M^-1) were investigated by an RIA using tritium-labeled 1, 25(OH)₂D₃. The data suggested that the rat monoclonal antibody 3R-1 derived from the hapten 3-HS and the mouse monoclonal antibody 24M-3 from the hapten 24-OA would be available for developing practical analytical systems.