Determination of glycated protein

Abstract

The present invention provides methods for quantitation of glycated protein in a biological sample using a solid support matrix by making a first bound protein measurement total bound protein under conditions where both glycated and non-glycated protein bind to the support in making a second bound protein measurement under conditions where glycated protein is bound to the support and non-glycated protein is not substantially bound. Diagnostic devices and kits comprising the methods of the present invention are also provided

1 . A diagnostic device, comprising:
 (1) a solid support matrix having a measurement area; and   (2) negatively charged groups and dihydroxyboryl groups immobilized on the solid support matrix, wherein said negatively charged groups are capable of binding both glycated and nonglycated protein at a first pH, and wherein said dihydroxyboryl groups are capable of binding glycated protein at a second pH.   
     
     
         2 . The device of  claim 1 , wherein the first pH is between about 5.0 and about 7.0. 
     
     
         3 . The device of  claim 1 , wherein the second pH is between about 8.0 and about 10.0. 
     
     
         4 . The device of  claim 1 , wherein the protein is hemoglobin. 
     
     
         5 . The device of  claim 1 , wherein the protein is albumin. 
     
     
         6 . The device of  claim 1 , wherein the dihydroxyboryl group is of the type 
       
         
           
           
               
               
           
         
         where R is selected from the group consisting of phenyl, alkyl of 1-6 carbons, ethyl, 1-propyl, 3-methyl-1-butyl and aminophenyl. 
       
     
     
         7 . The device of  claim 1 , wherein the solid support matrix is selected from the group consisting of cellulose, nitrocellulose, cellulose acetate, polyacrylamide, agarose polyacrylamide copolymer, agarose, starch, nylon, nylon polyesters, dextran, cross-linked dextran, dextran acrylamide copolymer, cross-linked hydroxyethylmethacrylate, substituted cross-linked polystyrenes, polyvinylalcohol, wool, metal oxides, porous ceramics coated with hydrophilic organic polymers and glass. 
     
     
         8 . A kit comprising the diagnostic device of  claim 1 , a first buffer configured to achieve the first pH, and a second buffer configured to achieve the second pH. 
     
     
         9 . The kit of  claim 8 , wherein the first pH is between about 5.0 and about 7.0. 
     
     
         10 . The kit of  claim 8 , wherein the second pH is between about 8.0 and about 10.0. 
     
     
         11 . The kit of  claim 8 , wherein the protein is hemoglobin. 
     
     
         12 . The kit of  claim 8 , wherein the protein is albumin. 
     
     
         13 . The kit of  claim 8 , wherein the dihydroxyboryl group is of the type 
       
         
           
           
               
               
           
         
         where R is selected from the group consisting of phenyl, alkyl of 1-6 carbons, ethyl, 1-propyl, 3-methyl-1-butyl and aminophenyl. 
       
     
     
         14 . The kit of  claim 8 , wherein the solid support matrix is selected from the group consisting of cellulose, nitrocellulose, cellulose acetate, polyacrylamide, agarose polyacrylamide copolymer, agarose, starch, nylon, nylon polyesters, dextran, cross-linked dextran, dextran acrylamide copolymer, cross-linked hydroxyethylmethacrylate, substituted cross-linked polystyrenes, polyvinylalcohol, wool, metal oxides, porous ceramics coated with hydrophilic organic polymers and glass. 
     
     
         15 . A method for determining a ratio of an amount of a glycated form of hemoglobin to a total amount of the hemoglobin in a blood sample, comprising:
 providing a solid support having negatively charged carboxyl groups immobilized thereon, which groups are capable of binding both the glycated and the nonglycated forms of the hemoglobin at a first pH, said support also having hydroxyboryl groups immobilized thereon, interspersed with the negatively charged carboxyl groups, which hydroxyboryl groups are capable of binding the glycated form of the hemoglobin at a second pH;   lysing the sample;   adding the sample to the solid support at the first pH, thereby binding both the glycated and the nonglycated forms of the hemoglobin to the negatively charged carboxyl groups on the solid support, and then performing a first measurement indicative of the total amount of the glycated and the nonglycated forms of the hemoglobin bound to the solid support;   changing the pH on the support to the second pH, thereby removing both the nonglycated form of the hemoglobin and the glycated form of the hemoglobin from the negatively charged carboxyl groups, after which removal the glycated form of the hemoglobin binds to the hydroxyboryl groups on the solid support independent of incubation time, and then performing a second measurement indicative of the amount of the glycated form of the hemoglobin bound to the solid support; and   determining the ratio of the amount of the glycated form of the hemoglobin to the total amount of the glycated and the nonglycated forms of the hemoglobin in the sample from the first and second measurements.   
     
     
         16 - 17 . (canceled) 
     
     
         18 . The method of  claim 15  further comprising adding a lysing agent to the solid support prior to adding the sample to the solid support. 
     
     
         19 - 20 . (canceled) 
     
     
         21 . A method for determining a ratio of an amount of a glycated form of a protein to a total amount of the protein in a sample containing both glycated and nonglycated forms of the protein, comprising:
 providing a solid support having negatively charged carboxyl groups immobilized thereon, which groups are capable of binding both the glycated and the nonglycated forms of the protein at a first pH, said support also having hydroxyboryl groups immobilized thereon, interspersed with the negatively charged carboxyl groups, which hydroxyboryl groups are capable of binding the glycated form of the protein at a second pH;   adding the sample to the solid support at the first pH, thereby binding both the glycated and the nonglycated forms of the protein to the negatively charged carboxyl groups on the solid support, and then performing a first measurement indicative of the total amount of the glycated and the nonglycated forms of the protein bound to the solid support;   changing the pH on the support to the second pH, thereby removing both the nonglycated form of the protein and the glycated form of the protein from the negatively charged carboxyl groups, after which removal the glycated form of the protein immediately binds to the hydroxyboryl groups on the solid support independent of incubation time, and then performing a second measurement indicative of the amount of the glycated form of the protein bound to the solid support; and   determining the ratio of the amount of the glycated form of the protein to total the total amount of the glycated and the nonglycated forms of the protein in the sample from the first and second measurements.   
     
     
         22 - 24 . (canceled) 
     
     
         25 . The method of  claim 21 , wherein the sample comprises blood, and wherein the protein comprises blood or albumin. 
     
     
         26 . A method for ascertaining the extent to which a protein in a sample has been glycated, comprising:
 providing a biological sample containing both glycated and nonglycated forms of a protein;   contacting the biological sample with a solid support at a first pH and binding both glycated and nonglycated forms of the protein to negatively charged groups immobilized on the solid support;   obtaining a first measurement indicative of the total amount of the glycated and nonglycated forms of the protein bound to the solid support at the first pH;   then changing the first pH on the solid support to a second pH, thereby removing both the glycated form of the protein and the nonglycated form of the protein from the negatively charged groups and immediately rebinding only the glycated form of the protein to hydroxyboryl groups immobilized on the solid support;   obtaining a second measurement indicative of the amount of the glycated form of the protein bound to the solid support at the second pH; and   relating the first measurement to the second measurement to ascertain the extent to which the protein in the sample has been glycated.   
     
     
         27 . The method of  claim 26  further comprising contacting the solid support with an aliquot of a buffer solution sufficient to rinse off excess biological sample, including both unbound glycated and nonglycated forms of the protein, prior to obtaining the first measurement. 
     
     
         28 - 32 . (canceled)