Rapid measurement of total vitamin d in blood

Abstract

Assays for rapid measurement of total vitamin D in blood are provided. Vitamin D is measured following the rapid and irreversible release of vitamin D due to denaturation and digestion of vitamin D binding proteins by aspartyl peptidases (e.g., pepsin) under acidic conditions. Such measurements may be made using a vitamin D binder (e.g., an antibody) to measure competition between free vitamin D and added, labeled vitamin D. Synergy between denaturation and degradation is believed to provide more rapid and more complete release of vitamin D than would occur with acid or enzyme alone. These measurements may be made using small amounts of whole blood, serum, or plasma, and are suitable for use in automated devices. These methods provide the advantages of reduced cost, increased speed, reduced discomfort to the subject, and increased availability and ease of use. Reagents, kits, devices, and systems for these assays are also disclosed.

1 - 61 . (canceled) 
     
     
         62 . A kit for determining the amount of vitamin D in a blood sample, the kit comprising:
 a low pH composition comprising an aspartyl peptidase active at low pH;   a neutral pH buffer; and   instructions for use for performing a method comprising:
 contacting said blood sample with said low pH composition comprising aspartyl peptidase active at low pH, whereby a low pH diluted blood sample composition is formed, effective to at least partially denature and at least partially digest a vitamin D binding protein in the blood sample; 
 contacting said low pH diluted blood sample composition with said neutral pH buffer, whereby a neutral pH diluted blood sample composition is formed, effective that said aspartyl peptidase is inactivated; and 
 measuring the amount of said vitamin D in said neutral pH diluted blood sample, wherein said measuring comprises using a competition assay measurement. 
   
     
     
         63 . The kit of  claim 62  further comprising a container for holding the low pH composition or the neutral pH buffer. 
     
     
         64 . The kit of  claim 63 , wherein said container comprises a septum, barrier, cap, or closure effective to prevent evaporation from the container. 
     
     
         65 . The kit of  claim 64 , wherein said septum, barrier, cap, or closure is effective to reduce or prevent loss of the low pH composition or the neutral pH buffer from said container. 
     
     
         66 . The kit of  claim 62 , wherein said low pH composition further comprises a buffer. 
     
     
         67 . The kit of  claim 66 , wherein said buffer comprises a buffer containing a citrate. 
     
     
         68 . The kit of  claim 62 , further comprising a vitamin D binder. 
     
     
         69 . The kit of  claim 68  wherein said instructions for use further includes instructions for contacting said neutral pH diluted blood sample composition with the vitamin D binder, wherein said vitamin D binder binds specifically to vitamin D, wherein said vitamin D binder is selected from an antibody, an antibody fragment, and an aptamer. 
     
     
         70 . The kit of  claim 62 , further comprising labeled vitamin D. 
     
     
         71 . The kit of  claim 70  wherein said instructions for use further includes instructions for adding said labeled vitamin D to said neutral pH diluted blood sample composition comprising vitamin D binder wherein said labeled vitamin D comprises a label selected from a dye, an epitope tag, a fluorescent moiety, a luminescent moiety, a chemiluminescent moiety, an enzymatic label, a magnetic label, a paramagnetic label, a contrast agent, a nanoparticle, a radioisotope, biotin, streptavidin, and a quencher. 
     
     
         72 . The kit of  claim 70 , wherein said labeled vitamin D comprises an alkaline phosphatase substrate or a horseradish peroxidase substrate. 
     
     
         73 . The kit of  claim 72  wherein said instructions for use further includes instructions for adding the alkaline phosphatase substrate or a peroxidase substrate. 
     
     
         74 . The kit of  claim 73  further comprising capture elements configured to bind to anti-vitamin D antibody, said capture elements are selected from a polynucleotide, a polypeptide, a peptide nucleic acid, a locked nucleic acid, an oligosaccharide, a polysaccharide, an antibody, an antibody fragment, an antibody mimic, an immunoadhesin, a receptor, a ligand, biotin, avidin, streptavidin, Extravidin, neutravidin, an avidin derivative, an avidin analog, a metal, and an epitope tag. 
     
     
         75 . The kit of  claim 73 , further comprising capture elements configured to bind to anti-vitamin D antibody, said capture elements is avidin, streptavidin, biotin, an avidin derivative, or an analog thereof. 
     
     
         76 . The kit of  claim 62  wherein said aspartyl peptidase comprises porcine gastric pepsin. 
     
     
         77 . The kit of  claim 62  wherein the buffer is effective to provide a pH of between about pH 2.5 and about pH 4 after sample addition. 
     
     
         78 . The kit of  claim 62  wherein said low pH comprises a pH of about pH 5 or less.