Methods and reagents for the rapid determination of glycated hemoglobin

A method for determining the relative amount of glycated hemoglobin in a whole blood sample containing glycated and non-glycated hemoglobin comprising:

• a. treating a whole blood sample to release glycated and non-glycated hemoglobin;
• b. diluting the sample to a final measurement concentration to form a diluted sample;
• c. combining a solid substrate having attached thereto a binding agent for glycated hemoglobin and the diluted sample;
• d. separating the solid substrate from the diluted sample immediately after combining the solid substrate and the diluted sample and subsequently measuring initial absorbance of the diluted sample;
• e. mixing the diluted sample and the solid substrate which was separated in step d under conditions sufficient to effect substantial binding of glycated hemoglobin to the solid substrate;
• f. separating the solid substrate from the diluted sample;
• g. measuring the absorbance of the diluted sample;
• and
• h. determining the relative amount of glycated hemoglobin in the whole blood sample from the initial absorbance measurement, the absorbance measurement in step g, and a standard curve prepared from one or more calibrators.

A method for determining the relative amount of glycated hemoglobin in a whole blood sample containing glycated and non-glycated hemoglobin comprising:

• a. treating a whole blood sample to release glycated and non-glycated hemoglobin;
• b. forming a diluted solution of the sample, said diluted solution of the sample having a final measurement concentration and containing MgSO4 and a solid substrate comprising particles having attached thereto a binding agent for glycated hemoglobin;
• c. separating the solid substrate from the diluted solution of the sample immediately after combining said solid substrate and said diluted sample and subsequently measuring initial absorbance of the diluted solution of the sample;
• d. mixing the diluted solution of the sample and the solid substrate which was separated in step c under conditions sufficient to effect substantial binding of glycated hemoglobin to the solid substrate;
• e. separating the solid substrate from the diluted solution of the sample;
• f. measuring absorbance of the diluted solution of the sample;
• and
• g. determining the relative amount of glycated hemoglobin in the whole blood sample from the initial absorbance measurement, the absorbance measurement in step f, and a standard curve prepared from one or more calibrators.

A kit for assaying the relative amount of glycated hemoglobin in a sample, said test kit comprising as separately packaged components:

• a. a reagent solution containing a buffer, about 10 mM to about 500 mM MgSO.sub.4, and a solid substrate having attached thereto a binding agent for glycated hemoglobin;
• b. a diluent buffer solution which contains at least one proteinaceous stabilizer selected from the group consisting of gelatin, polyvinylpyrrolidone, casein and albumin and which is capable of maintaining pH at a value in the range between about 7.6 and 9.6;
• and
• c. an agent for releasing and non-glycated hemoglobin from a whole blood sample.

A method for determining the relative amount of glycated hemoglobin in a whole blood sample containing glycated and non-glycated hemoglobin comprising the following sequential steps:

• a. treating a whole blood sample to release, glycated and non-glycated hemoglobin;
• b. diluting the sample to a final measurement concentration to form a diluted sample;
• c. combining a solid substrate having attached thereto a binding agent for glycated hemoglobin and the diluted sample;
• d. measuring initial absorbance of the diluted sample immediately after combining the solid substrate and the diluted sample;
• e. mixing the diluted sample and the solid substrate under conditions sufficient to effect substantial binding of glycated hemoglobin to the solid substrate;
• f. separating the solid substrate from the diluted sample;
• and
• g. determining the amount of glycated hemoglobin bound by the solid substrate by measuring the absorbance of the diluted sample.