Monoclonal antibody for calcitriol (l α,25-dihydroxyvitamin d3)

Hybridoma cell lines secreting antibodies for vitamin D3 metabolites have been generated by fusing splenocytes from BALB/c mice immunized with 3β-glutaryl-25-hydroxyvitamin D_s conjugated to bovine serum albumin (3β-glu-25-OH-D_s-BSA) and Sp2/O-Agl4 myeloma cells. Purification of monoclonal antibodies from culture media or ascites fluids was accomplished by procedures including affinity chromatog-raphy on Protein A-Sepharose 4B. Each monoclonal antibody was analyzed as to its affinity and specificity by equilibrium dialysis and an enzyme immunoassay (EIA) based on a double antibody system. It was demonstrated that clone 1C2-60 pro-duced an antibody highly specific to la,25-dihydroxyvitamin D3 (calcitriol), and the clone 2B3-66 antibody was reactive to 25-hydroxyvitamin D3 and similar structural compounds. These two monoclonal antibodies produced by 1C2-60 and 2B3-66 were determined to belong to the IgG2a class, and their affinity constants (K_a) with 3β-glu-25-OH-D_s were demonstrated to be 3.6×10⁹M^-1 and 2.9×10⁹M^-1, respectively, at 4°C. The characteristics of these monoclonal antibodies were compared with those of conventional antibodies raised in mice and rabbits. Finally, by using monoclonal antibody 1C2-60, a sensitive EIA has been developed that can detect 10 pg of calcitriol.