Measurement of plasma 1,25 dihydroxyvitamin D using a novel immunoextraction technique and immunoassay with iodine labelled vitamin D tracer

We evaluated a novel assay for the measurement of 1,25 dihydroxyvitamin D (1,25(OH)(2)D). Immunoextraction of 1,25 (OH)(2)D is performed using a mini column containing a solid-phase monoclonal antibody followed by radio-immunoassay (RIA) using an I-125-labelled 1,25 (OH)(2)D derivative tracer and Sac-cell separation. The mean recovery of 1,25(OH)(2)D-3 was 101%. linearity was excellent. inter- and intra-assay coefficients of variation were 9.8 and 13% and 11, 10 and 14% at low, medium and high concentrations of 125(OH)(2)D-3, respectively. The crossreactivity of vitamin D metabolites was < 0.0015% for 25-hydroxyvitamin D-3, 24,25 dihydroxyvitamin D-3 and dihydrotachysterol and 0.54% for 1 alpha calcidol, 1,25 dihydroxyvitamin D-2 cross-reactivity was 79%. The detection limit of the assay was 5 pmol/L. Comparison with a commercial radio receptor assay (RRA) and an in-house RIA gave regression equations of y = 0.94x + 11.8 (r = 0.98) and y = 0.91x - 1.7 (r = 0.95), respectively, with no major discrepancies between the methods in all patient groups studied. Plasma concentrations of 1.25(OH)(2)D obtained with the assay were as follows: normal, unsupplemented subjects: mean 88, range 48-155 pmol/L, n=68. patients with chronic renal failure: mean 11, range 3-36 pmol/L, n = 27, primary hyperparathyroidism: mean 198, range 130-299 pmol/L, n = 23. Paget's disease: mean 92, range 42-149 pmol/L, n=24, osteomalacia: mean 43, range 27-61 pmol/L, n=9. A minimum sample Volume of 300 mu L is required, the hands-on rime is significantly less than other commercial assays and the measuring procedure is gamma counting rather than scintillation counting, The assay offers several advantages over previous methods and should allow more laboratories to offer measurement of 1,25 (OH)(2)D as part of their repertoire.