Methods and compositions for assaying vitamin d

Abstract

The present invention provides methods and compositions, e.g., kits, for assaying a vitamin D moiety in a sample, comprising or using, inter alia, a buffer that is capable of dissociating a vitamin D moiety from its binding protein and/or a buffer of acidic pH, and at least two antibodies, e.g., at least two monoclonal antibodies, that are separately conjugated to particles, e.g., latex particles, wherein at least one of said antibodies (or the first antibody) has a specific binding affinity towards the vitamin D moiety, and at least another said antibody (or the second antibody) has a specific binding affinity towards the complex formed between the first antibody and the vitamin D moiety, if present in said sample. In some embodiments, the optical change due to the agglutination reaction between the antibodies and the vitamin D moiety is measured for determination of the amount of vitamin D content in the samples. Kits and reaction mixtures for assaying a vitamin D moiety in a sample are also provided.

1 - 35 . (canceled) 
     
     
         36 . A method for assaying a vitamin D moiety in a sample, which method comprises:
 a) contacting said sample with a first reagent comprising a buffer of acidic pH to dissociate said vitamin D moiety in said sample from its binding proteins;   b) contacting said vitamin D moiety in said sample from step a) with a second reagent comprising particles, e.g., magnetic particles, coated with a first antibody that has a specific binding affinity to said vitamin D moiety to form a vitamin D moiety/first antibody complex; and   c) contacting said vitamin D moiety/first antibody complex with a third reagent comprising a second antibody labelled with a signal generating moiety or molecule to form a complex among said vitamin D moiety, said first antibody and said second antibody labelled with a signal generating moiety or molecule; and   d) assessing said signal from said complex among said vitamin D moiety, said first antibody complex and said second antibody to determine the presence, absence and/or amount of the vitamin D moiety in said sample.   
     
     
         37 . The method of  claim 36 , wherein the signal generating moiety or molecule is selected from the group consisting of acridinium ester, isoluminol, alkaline phosphatase, horse radish peroxidase and fluoresin. 
     
     
         38 - 39 . (canceled) 
     
     
         40 . The method of  claim 36 , which does not comprise a step of removing a natural vitamin D binding protein from the sample prior to assessing binding between the antibodies and the vitamin D moiety. 
     
     
         41 . The method of  claim 36 , wherein the pH in a final reaction mixture comprising the sample, the acidic pH buffer and antibodies conjugated on particles, e.g., nanoparticles, ranges from about 3.0 to about 13, preferably from about 5.5 to about 8.5. 
     
     
         42 . The method of  claim 36 , which is used to assess status of the vitamin D moiety in a subject, and the sample is a biological sample obtained and/or derived from the subject. 
     
     
         43 . The method of  claim 36 , wherein the vitamin D moiety is 25(OH)D2,  25 (OH)D3 or a sum of 25(OH)D2 and 25(OH)D3. 
     
     
         44 . The method of  claim 36 , wherein the acidic pH buffer is the sodium acetate buffer, or sodium citrate buffer, or phosphoric acid buffer, a combination thereof, or any buffer having a buffer function in the acidic pH range. 
     
     
         45 . The method of  claim 44 , wherein the pH of the acidic buffer is in the range from about 2.5 to about 6.9. 
     
     
         46 . The method of  claim 36 , wherein the antibodies are paired monoclonal antibodies with one antibody or the first antibody having a specific binding affinity towards the vitamin D moiety, and the other antibody or the second antibody having a specific binding affinity towards the complex formed between the first antibody and the vitamin D moiety. 
     
     
         47 . The method of  claim 46 , wherein the first antibody specifically binds to 25(OH)D, and the second antibody specifically binds to the complex formed between the first antibody and 25(OH)D. 
     
     
         48 . The method of  claim 36 , wherein the acidic pH buffer contains a salt, polymer, and/or a detergent that allows for partial or total dissociation of vitamin D moiety from its binding protein(s) within a short period of time (e.g., less than 30 min). 
     
     
         49 . The method of  claim 48 , wherein the salt, polymer, and/or a detergent is included in the acidic pH buffer, or in a separated reagent solution used to mix with the acidic pH buffer in the assay procedure. 
     
     
         50 . The method of  claim 36 , wherein the particles, e.g., nanoparticles, comprise polystyrene, polymethyl methacrylate, polymethyl naphthalene, poly(divinylbenzene), polyvinyl naphthalene, co-polymer of styrene, acrylic acid divinylbenzene, naphathalene, carbon 60, magnetic beads, gold, silver, silica, silicon dioxide, chromium dioxide, and/or titanium dioxide. 
     
     
         51 . The method of  claim 36 , which is conducted using a magnetic particle (bead) based immunoassay method. 
     
     
         52 . The method of  claim 51 , wherein the particle sizes (diameters) can range from about 500 nm to about 2,000 nm, optionally the first antibody is coated on the magnetic particles and the second antibody is labeled with chemiluminescent or fluorescent signal generating molecules such as acridinium ester, isoluminol, alkaline phosphatase and horse radish peroxidase, or fluroresin or green proteins, and further optionally the assay is conducted in a heterogeneous format involving one or two washing steps. 
     
     
         53 . The method of  claim 52 , wherein the assay is a heterogeneous assay involving phase separation step or steps. 
     
     
         54 . The method of  claim 36 , wherein the assay is completed within 30 min or less. 
     
     
         55 . The method of  claim 36 , wherein the assay is conducted with a general chemistry analyzer or a clinical chemistry analyzer.