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 - 40 . (canceled) 
     
     
         41 . 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.   
     
     
         42 . The method of  claim 41 , wherein the sample is lysed by a lysing agent. 
     
     
         43 . The method of  claim 42 , wherein the lysing agent comprises Triton X-100 or Igepal CA-630. 
     
     
         44 . The method of  claim 42  further comprising adding the lysing agent to the solid support prior to adding the sample to the solid support. 
     
     
         45 . The method of  claim 42 , wherein the first buffer comprises the lysing agent. 
     
     
         46 . The method of  claim 41 , wherein the solid support 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. 
     
     
         47 . 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 said dihydroxyboryl groups are capable of binding glycated protein at a second pH.   
     
     
         48 . The device of  claim 47 , wherein the first pH is between about 5.0 and about 7.0. 
     
     
         49 . The device of  claim 47 , wherein the second pH is between about 8.0 and about 10.0. 
     
     
         50 . The device of  claim 47 , wherein the protein is hemoglobin. 
     
     
         51 . The device of  claim 47 , wherein the protein is albumin. 
     
     
         52 . The device of  claim 47 , 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. 
       
     
     
         53 . The device of  claim 47 , 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. 
     
     
         54 . A kit comprising the diagnostic device of  claim 47 , a first buffer configured to achieve the first pH, and a second buffer configured to achieve the second pH. 
     
     
         55 . The kit of  claim 54 , wherein the first pH is between about 5.0 and about 7.0. 
     
     
         56 . The kit of  claim 54 , wherein the second pH is between about 8.0 and about 10.0. 
     
     
         57 . The kit of  claim 54 , wherein the protein is hemoglobin. 
     
     
         58 . The kit of  claim 54 , wherein the protein is albumin. 
     
     
         59 . The kit of  claim 54 , 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. 
       
     
     
         60 . The kit of  claim 54 , 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.