Vitamin d assays

Abstract

The disclosure relates to methods for measuring levels of 25(OH)vitamin D (25OHD) in a mammalian fluid sample. The disclosure further relates to kits for measuring levels of 25(OH)vitamin D (25OHD) in a mammalian fluid.

1 . A method for measuring 25(OH)vitamin D (25OHD) in a mammalian fluid sample comprising the steps of:
 a. contacting a sample containing 25OHD) with an antibody that specifically binds to 25OHD in the presence of a release reagent; and   b. measuring the amount of 25OHD bound to the antibody.   
     
     
         2 . The method of  claim 1 , wherein the release reagent is selected from 1-butyl-4-methylpyridinium chloride, 1-ethyl-3-methylpyridinium ethyl sulfate, and a mixture thereof. 
     
     
         3 . The method of  claim 1 , wherein the release reagent further comprises methyl β-cyclodextrin, sodium salicylate or a mixture thereof. 
     
     
         4 . The method of  claim 1 , wherein the release reagent comprises 1-butyl-4-methylpyridinium chloride, 1-ethyl-3-methylpyridinium ethyl sulfate, methyl β-cyclodextrin, and sodium salicylate. 
     
     
         5 . The method of  claim 1 , wherein the antibody is selected from a monoclonal antibody, a polyclonal antibody, an antigen binding fragment of a monoclonal antibody, and an antigen binding fragment of a polyclonal antibody. 
     
     
         6 . The method of  claim 1 , wherein the antibody is in solution. 
     
     
         7 . The method of  claim 1 , wherein the antibody is bound to a surface. 
     
     
         8 . The method of  claim 7 , wherein the antibody is bound to said surface directly, 
     
     
         9 . The method of  claim 7 , wherein the antibody is bound to said surface indirectly. 
     
     
         10 . The method of  claim 9 , wherein the antibody is bound to said surface by a secondary antibody that specifically binds to said antibody. 
     
     
         11 . The method of  claim 1 , in which the 25OHD is a conjugate, and which comprises before step (a) a step of binding said 25OHD conjugate to a surface. 
     
     
         12 . The method of  claim 1 , which comprises after step (a) a step of binding 25OHD to a surface, wherein the 25OHD is bound to said surface by the antibody that specifically binds to 25OHD. 
     
     
         13 . The method of  claim 1 , which comprises before step (a) a step of binding 25OHD) to a surface, wherein the 25OHD is bound to said surface indirectly by an antibody that specifically recognizes the antibody that specifically binds to 25OHD. 
     
     
         14 . The method of  claim 1 , wherein the 25OHD is bound to an enzyme. 
     
     
         15 . The method of  claim 1 , wherein the 25OHD is bound to a substrate of an enzyme. 
     
     
         16 . The method of  claim 14 , which comprises before step (h) a step of adding a substrate of said enzyme to the mixture of step (a), wherein the action of said enzyme on the substrate produces a signal that is measured in step (b). 
     
     
         17 . The method of  claim 15 , which comprises before step (b) a step of adding an enzyme to the mixture of step (a), wherein said enzyme recognizes the substrate bound to the 25OHD, and wherein the action of said enzyme on the substrate produces a signal that is measured in step (h). 
     
     
         18 . The method of  claim 1 , wherein in step (a) said contacting is performed in the presence of add-in 25OHD bound to an enzyme. 
     
     
         19 . The method of  claim 1 , which comprises after step (a) a step of adding add-in 25OHD bound to an enzyme to the mixture of step (a), wherein the action of the enzyme on a substrate bound to the antibody produces a signal that is measured in step (b), and wherein the signal measured in step (b) is inversely proportional to the amount 25OHD in the sample. 
     
     
         20 . The method of  claim 1 , which comprises after step (a) a step of adding add-in 25OHD bound to a substrate of an enzyme to the mixture of step (a), wherein the action of the enzyme bound to the antibody on the substrate produces a signal that is measured in step (b), and wherein the signal measured in step (b) is inversely proportional to the amount of 25OHD in the sample. 
     
     
         21 . A method of measuring 25(OH)vitamin D (25OHD) in a mammalian fluid sample comprising the steps of:
 a. contacting a sample comprising 25OHD with a dissociation buffer comprising a release reagent selected from 1-butyl-4-methylpyridinium chloride, 1-ethyl-3-methylpyridinium ethyl sulfate and a mixture of 1-butyl-4-methylpyridinium chloride and 1-ethyl-3-methylpyridinium ethyl sulfate in a well of a microtiter plate;   b. contacting the mixture of step (a) with (i) add-in 25OHD conjugated to an enzyme and an antibody that specifically binds to 25OHD;   c. washing the well to remove add-in 25OHD that is not specifically bound;   d. adding a substrate of said enzyme conjugated to add-in 25OHD;   e. quantitatively measuring the signal produced by the reaction of substrate and enzyme in the well; and   f. comparing the measured signal with a standard curve of measurements of the product with known quantities of 25OHD 3 ,25OHD 2  or a mixture thereof, to determine the amount of 25OHD in the sample.   
     
     
         22 . The method of  claim 21 , wherein the dissociation buffer comprises 1-butyl-4-methylpyridinium chloride and 1-ethyl-3-methylpyridinium ethyl sulfate. 
     
     
         23 . The method of  claim 22 , wherein the release reagent further comprises methyl β-cyclodextrin and sodium salicylate. 
     
     
         24 . The method of  claim 21 , wherein the antibody is selected from a monoclonal antibody, a polyclonal antibody, an antigen binding fragment of a monoclonal antibody, and an antigen binding fragment of a polyclonal antibody. 
     
     
         25 . The method of  claim 21 , wherein the antibody is in solution. 
     
     
         26 . The method of  claim 22 , wherein the antibody is bound to the surface of the well. 
     
     
         27 . The method of  claim 26 , wherein the antibody is bound to said surface directly. 
     
     
         28 . The method of  claim 26 , wherein the antibody is bound to said surface indirectly. 
     
     
         29 . The method of  claim 28 , wherein the antibody is bound to said surface by a secondary antibody that specifically binds to said antibody. 
     
     
         30 . A kit for measuring 25(OH)vitamin D (25OHD) in a mammalian fluid sample comprising
 a. an antibody that specifically binds to 25OHD; and   b. a dissociation buffer comprising a release reagent selected from 1-butyl-4-methylpyridinium chloride, 1-ethyl-3-methylpyridinium ethyl sulfate, and a mixture thereof.   
     
     
         31 . The kit of  claim 30 , wherein the dissociation buffer comprises 1-butyl-4-methylpyridinium chloride and 1-ethyl-3-methylpyridinium ethyl sulfate. 
     
     
         32 . The kit of  claim 31 , wherein the dissociation buffer further comprises methyl 3-cyclodextrin and sodium salicylate. 
     
     
         33 . The kit of  claim 30 , which further comprises a microtiter plate with a well that is coated with an antibody. 
     
     
         34 . The kit of  claim 33 , wherein the well is coated with a secondary antibody that specifically binds to said antibody that specifically binds to 25OHD. 
     
     
         35 . The kit of  claim 33 , wherein the microtiter plate is coated with the antibody that specifically binds to 25OHD. 
     
     
         36 . The kit of  claim 30 , wherein the mammalian fluid sample is serum or plasma. 
     
     
         37 . The kit of  claim 30 , which further comprises a conjugate of an enzyme with add-in 25OHD. 
     
     
         38 . The kit of  claim 37 , wherein the enzyme is conjugated to add-in 25OHD via an N-hydroxysuccinimide ester. 
     
     
         39 . The kit of  claim 38 , wherein the enzyme is alkaline phosphatase.