Selection of high-affinity and high-specificity monoclonal antibodies for 1α,25-dihydroxyvitamin D

The preparation of high-affinity and high-specificity monoclonal antibodies to 1α,25-dihydroxyvitamin D is described. Monoclonal antibodies were derived from Balb-c mice immunised with either 1α-hydroxy-25,26,27-trinor-24-cholecalcioic acid or with 1α-hydroxy-26,27-dinor-chole-calciferol-25-oxime, and spleen cells were hybridised with mouse myeloma cells. From six fusions nine monoclonal antibodies (MAb's) were selected from 676 antibody-secreting hybrids. Antibodies varied widely in their ability to bind 1α,25-dihydroxyvitamin D₃ (50% displacement of radioligand ranged from 25 - 900 pg); two had particularly useful characteristics for 1α,25-dihydroxyvitamin D assay. MAb 5F2 has high affinity (Ka = 1.39 × 10¹⁰M^-1) and does not discriminate between 1α,25-dihydroxyvitamin D₂ and D₃, thus enabling the two forms to be measured together. MAb 1G7 is highly specific, having no cross-reactivity with 25-hydroxy-, 24,25-dihydroxy- or 25,26-dihydroxyvitamin D at concentrations found in normal human serum; this MAb has the potential to eliminate the lengthy extraction procedures involved in currently available assays for 1α,25-dihydroxyvitamin D.