Rapid measurement of total vitamin D in blood
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.
1. A method for determining the amount of vitamin D in a blood sample, wherein said blood sample comprises vitamin D and a vitamin D binding protein, the method comprising:
contacting said blood sample with a low pH composition comprising an 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 said vitamin D binding protein;
contacting said low pH diluted blood sample composition with a neutral pH composition comprising a 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 a competition assay measurement.
2. The method of claim 1 , wherein said low pH composition comprising an aspartyl peptidase active at low pH comprises a buffer.
3. The method of claim 2 , wherein said buffer comprises a buffer containing a citrate.
4. The method of claim 1 , contacting said neutral pH diluted blood sample composition with a 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.
5. The method of claim 1 further comprising:
adding labeled vitamin D to said neutral pH diluted blood sample composition comprising said 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.
6. The method of claim 5 , wherein said labeled vitamin D comprises an alkaline phosphatase label or a horseradish peroxidase label.
7. The method of claim 6 further comprising:
contacting said neutral pH diluted blood sample composition comprising said vitamin D binder with a capture surface, wherein said capture surface comprises capture elements configured to bind to said vitamin D binder; and
detecting said labeled vitamin D, wherein the amount of detected vitamin D provides a measure of the amount of vitamin D in the blood sample
wherein detecting said labeled vitamin D comprises adding an alkaline phosphatase substrate or a peroxidase substrate.
8. The method of claim 7 , wherein said capture elements configured to bind to said anti-vitamin D antibody 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.
9. The method of claim 8 , wherein said capture elements configured to bind to said anti-vitamin D antibody comprise avidin, streptavidin, biotin, an avidin derivative, or an analog thereof.
10. The method of claim 1 , wherein said blood sample has a volume of less than about 25 μL.
11. The method of claim 1 , wherein said blood sample has a volume of less than about 10 μL.
12. The method of claim 1 , wherein said blood sample comprises a blood sample selected from a sample of whole blood, a sample of blood plasma, and a sample of blood serum.
13. The method of claim 1 wherein said aspartyl peptidase comprises porcine gastric pepsin.
14. The method of claim 2 wherein the buffer effective to provide a pH of between about pH 2.5 and about pH 4 after sample addition.
15. A method for determining the amount of vitamin D in a blood sample, wherein said blood sample comprises vitamin D and a vitamin D binding protein, the method comprising:
contacting said blood sample with a low pH composition comprising an 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 said vitamin D binding protein;
contacting said low pH diluted blood sample composition with a neutral pH composition comprising a 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 a competition assay measurement;
wherein said low pH comprises a pH of about pH 5 or less.
16. A method for determining the amount of vitamin D in a blood sample, wherein said blood sample comprises vitamin D and a vitamin D binding protein, the method comprising:
contacting said blood sample with a low pH composition comprising an 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 said vitamin D binding protein;
contacting said low pH diluted blood sample composition with a neutral pH composition comprising a 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 a competition assay measurement;
performing another assay for the measurement of another analyte in the blood sample.