Methods and reagents for determining isomeric analytes

Abstract

Methods include determining in a sample an amount of a first isomeric analyte and a second isomeric analyte. A first measurement value and a second measurement value are determined. The first measurement value represents a total amount of the first isomeric analyte and the second isomeric analyte. The second measurement value represents an amount of the second isomeric analyte only. The second measurement value is subtracted from the first measurement value to obtain a resulting value and the resulting value is equated to an amount of the first isomeric analyte in the sample.

1 - 20 . (canceled) 
     
     
         21 . A method of determining in a sample an amount of non-epi-25-hydroxy vitamin D 3  and 3-epi 25-hydroxy vitamin D 3 , the method comprising:
 (a) conducting an assay on a first portion of the sample using an assay protocol wherein assay reagents utilized in the assay protocol of this step (a) comprise vitamin D 3  conjugated to a label and a first antibody having a binding affinity for each of non-epi-25-hydroxy vitamin D 3  and 3-epi 25-hydroxy vitamin D 3  of 10 8  to 10 14  liters/mole to form a first complex comprising the first antibody and non-epi-25-hydroxy vitamin D 3  and a second complex comprising the first antibody and 3-epi 25-hydroxy vitamin D 3  wherein the first complex and the second complex include vitamin D 3  conjugated to a label wherein an amount of signal from the first complex and the second complex is related to a total amount non-epi-25-hydroxy vitamin D 3  and 3-epi 25-hydroxy vitamin D 3  in the sample to obtain a first measurement value; and   (b) conducting the assay on a second portion of the sample using the same assay protocol as in step (a) wherein assay reagents utilized in the assay protocol in this step (b) comprise the vitamin D 3  conjugated to a label and the first antibody, wherein a second antibody having a binding affinity for the non-epi-25-hydroxy vitamin D 3  of 10 6  to 10 8  liters/mole and a binding affinity for the 3-epi 25-hydroxy vitamin D 3  of less than about 10 4  liters/mole is employed in an amount of about 5 to about 200 times the amount of the first antibody, wherein the binding affinity of the second antibody for non-epi-25-hydroxy vitamin D 3  is less than the binding affinity of the first antibody for non-epi-25-hydroxy vitamin D 3  by a factor of at least about 10, wherein the second antibody binds the non-epi-25-hydroxy vitamin D 3  such that the non-epi-25-hydroxy vitamin D 3  does not bind to the first antibody, and wherein a complex is formed comprising the first antibody and the epi-25-hydroxy vitamin D 3 , wherein the complex includes the vitamin D 3  conjugated to a label wherein an amount of signal from the complex is related to an amount of the 3-epi 25-hydroxy vitamin D 3  in the sample to obtain a second measurement value;   
       wherein an amount of the non-epi-25-hydroxy vitamin D 3  in the sample is determined by subtracting the second measurement value from the first measurement value. 
     
     
         22 . The method according to  claim 21 , wherein the assay protocol is a competitive homogeneous assay protocol. 
     
     
         23 . The method according to  claim 21 , wherein the assay protocol employs reagents that comprise a particle. 
     
     
         24 . The method according to  claim 21 , wherein the assay protocol employs reagents that comprise a photosensitizer reagent and a chemiluminescent particle. 
     
     
         25 . The method according to  claim 24 , wherein the photosensitizer reagent comprises a particle.