QUANTIFICATION OF CIRCULATING 1,25-DIHYDROXYVITAMIN-D BY RADIOIMMUNOASSAY WITH AN I-125 LABELED TRACER

We report here the first RIA for 1,25-dihydroxyvitamin D utilizing a r adioiodinated (I-125) tracer. This is also the first validated RIA for 1,25-dihydroxyvitamin D [1,25(OH)(2)D] that does not require sample p repuriiication by HPLC before the binding assay. The assay involves ac etonitrile extraction, treatment of the crude extract supernate with s odium periodate, extraction and purification of endogenous 1,25(OH)(2) D by solid-phase chromatography, and finally, quantification by RIA. C alibrators were prepared in stripped human serum and processed exactly the same as samples, eliminating the need for internal control for pr ocedural losses of endogenous 1,25(OH)(2)D. The assay consists of a 2- h room temperature incubation with the primacy antibody, a 20-min incu bation with a second antibody, and separation of bound from free by ce ntrifugation. Assay results can be in hand within 5 h. The detection l imit of the assay is 2.4 ng/L 1,25-dihydroxyvitamin D-3. Results compa re well with those from an accepted radioreceptor assay. Sample pretre atment with sodium periodate is absolutely essential before quantifica tion by RIA; otherwise, concentrations of endogenous 1,25(OH)(2)D may be greatly overestimated.