The present disclosure generally relates to assays for vitamin D metabolites. More specifically, assays are provided that utilize reagents that adequately release the metabolite 25(OH)vitamin D2 and 25(OH)vitamin D3 from vitamin D binding protein (DBP), where the release reagents are identified that do not interfere with the assay.
Assays for metabolically active vitamin D in samples such as mammalian fluids (including milk, whole blood, serum and plasma) generally measure quantities of both 25(OH)vitamin D2 (25OHD2) and 25(OH)vitamin D3 (25OHD3) (collectively, 25OHD). 25OHD2 is metabolically converted from vitamin D2 that is found in foods, and 25OHD3 is metabolically converted from vitamin D3 that is present in some foods and the result of photolytic conversion of 7-dehydrocholesterol by sunlight (Dusso et al. 2005. Vitamin D. Am J Physiol Renal Physiol 289:F8; U.S. Pat. No. 8,003,400; U.S. Pat. No. 7,087,395). These 25OHD assays generally utilize chromatographic procedures since most immunoassays, when not preceded by laborious extraction and reconstitution procedures, have inaccuracies that are dependent on the DBP concentration (Heijboer et al. 2012. Clin. Chem. 58: 543-548). Furthermore, reagents that release 25OHD from DBP (“release reagents”) can interfere with immunoassays but not chromatographic procedures (U.S. Pat. No. 8,003,400).
In humans, the DBP circulates at a 20-100 fold greater molar excess as compared to 25OHD (Dusso 2005). Because of this ratio, an effective immunoassay protocol must not simply be able to dissociate bound 25OHD from DBP but must also block the remaining available DBP so the analyte is not re-bound, an endpoint whose difficulty is magnified due to DBP having the greatest affinity towards the 25(OH) metabolite of vitamin D. In addition, reagents that release 25OHD from DBP will also act to release 25OHD from other proteins, such a 25OHD-specific antibody utilized in an immunoassay. Also, vitamin D is by nature a hydrophobic molecule so therefore once it is freed from DBP it must be in an environment in which it will remain free and therefore available for detection in the immunoassay. As such, a reagent used to dissociate bound 25OHD from DBP (“dissociation buffer”) in an immunoassay must provide for the release of the majority of the sample 25OHD from DBP such that the 25OHD is available for binding to the anti-25OHD antibody while not interfering with the binding of 25OHD to the specific antibody utilized in the immunoassay.
There is, therefore, a need for an immunoassay that overcomes the above obstacles to provide a simplified, more rapid and more accurate assay than previous assays for 25OHD.
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USPTO Backgrounds