DETERMINATION OF GLYCATED PROTEIN
First Claim
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1. 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 support at a first pH and binding both glycated and nonglycated forms of the protein to negatively charged groups immobilized on the support;
obtaining a first measurement reflective of the total amount of the glycated and nonglycated forms of the protein bound to the support at the first pH;
then changing the 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 support;
obtaining a second measurement reflective of the amount of the glycated form of the protein bound to the 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.
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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
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Citations
29 Claims
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1. A method for ascertaining the extent to which a protein in a sample has been glycated, comprising:
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providing a biological sample containing both glycated and nonglycated forms of a protein;
contacting the biological sample with a support at a first pH and binding both glycated and nonglycated forms of the protein to negatively charged groups immobilized on the support;
obtaining a first measurement reflective of the total amount of the glycated and nonglycated forms of the protein bound to the support at the first pH;
then changing the 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 support;
obtaining a second measurement reflective of the amount of the glycated form of the protein bound to the 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method for ascertaining the extent to which albumin in a sample has been glycated, comprising:
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providing a strip-type 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 albumin at a first pH between about 5.0 and about 7.0, and said dihydroxyboryl groups are capable of binding glycated albumin at a second pH between about 8.0 and about 10.0;
adding the sample to the solid support matrix at the first pH, thereby binding both glycated and nonglycated albumin to the negatively charged groups on the solid support matrix, and then performing a first measurement on the measurement area indicative of the total amount of glycated and nonglycated albumin bound to the solid support matrix;
changing the pH on the solid support matrix to the second pH, thereby removing both the nonglycated albumin and the glycated albumin from the negatively charged groups, after which removal the glycated albumin immediately binds to the dihydroxyboryl groups on the solid support matrix independent of incubation time, and then performing a second measurement on the measurement area indicative of the amount of glycated albumin bound to the solid support matrix; and
relating the first measurement to the second measurement to ascertain the extent to which the albumin in the sample has been glycated. - View Dependent Claims (17, 18, 19, 20, 21, 22)
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23. A method for ascertaining the extent to which hemoglobin in a sample has been glycated, comprising:
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providing a strip-type 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 hemoglobin at a first pH between about 5.0 and about 7.0, and said dihydroxyboryl groups are capable of binding glycated hemoglobin at a second pH between about 8.0 and about 10.0;
adding the sample to the solid support matrix at the first pH, thereby binding both glycated and nonglycated hemoglobin to the negatively charged groups on the solid support matrix, and then performing a first measurement on the measurement area indicative of the total amount of glycated and nonglycated hemoglobin bound to the solid support matrix;
changing the pH on the solid support matrix to the second pH, thereby removing both the nonglyeated hemoglobin and the glycated hemoglobin from the negatively charged groups, after which removal the glycated hemoglobin immediately binds to the dihydroxyboryl groups on the solid support matrix independent of incubation time, and then performing a second measurement on the measurement area indicative of the amount of glycated hemoglobin bound to the solid support matrix; and
relating the first measurement to the second measurement to ascertain the extent to which the hemoglobin in the sample has been glycated. - View Dependent Claims (24, 25, 26, 27, 28, 29)
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Specification