Method and reagent for determining vitamin d metabolites

[0074] Embodiment 1: Preparation of vitamin D releasing agent

[0075] Sodium toluenesulfonate was dissolved in water and adjusted to a final concentration of 1M sodium toluenesulfonate, 100 mM sodium citrate, and 50 mM iron(III) chloride with a stock solution of 1 M sodium citrate and 1 M iron(III) chloride.

[0076] Example 2: Binding of 25-OHD antibody on solid phase

[0077] Antibodies to 25-OH-vitamin D3 or 25-OH-vitamin D2 were diluted to a concentration of 1 μg / ml in Tris salt buffer at pH 8.0. The cavity of a microtiter plate (Maxisorb, Nunc) was coated with 100 μl of antibody dilution, dried at 37°C and left at 4°C until assayed.

[0078] Example 3: Competitive binding assay

[0079] Serum matrix without vitamin D (Seracon, Seracare Inc.) had been mixed with the established 25-OHD3 concentrations. 80 μl of each concentration series were filled into microtiter plate cavities pre-coated with 25-OHD-biotin tracer and incubated for 5 minutes at RT (20-27° C.) to allow tracer resolubility. Next, 80 [mu]l of the vitamin D releasing agent of Example 1 was added to the sample and mixed. 100 μl of the sample diluted with vitamin D releaser was transferred into the 25OHD-antibody coated cavity and incubated at room temperature for 30 minutes. Next, the sample mixture was removed and the cavities were washed 3 times with 200 μl each of wash buffer. The biotin-25OHD tracer bound to the antibody was detected by means of a peroxidase-labeled streptavidin and tetramethylbenzidine (TMB)-chromogenic reaction. Optical density was measured at 450 nm after the substrate reaction was stopped by adding 100 μl of 50 mM phos...