Measurement of glycosylated hemoglobin by immunoassay
First Claim
1. A method for determining the percentage of glycosylated hemoglobin fraction HbAlc in the total hemoglobin for a blood sample comprising:
- (a) preparing a mixture by contacting a blood smaple in a diluent with a monoclonal antibody specific for the glycoside portion of HbAlc and an antibody specific for the here portion of total hemoglobin whereby HbAlc in said sample binds to said monoclonal antibody to give a first bound fraction and nonglycosylated hemoglobin in said sample binds to said antibody to give a second bound fraction;
(b) passing a first beam of light having a wavelength maximum of about 416 nm through said mixture, whereby said first and second bound fractions absorb said ligth to give a first absorbance due to said total hemoglobin;
(c) measuring said first absorbance;
(d) adding to said mixture a source of peroxide and a peroxidase substrate whereby peroxide from said source reacts with said substrate to give a product, said reaction being catalyzed by HbAlc in said first bound fraction but not by nonglycosylated hemoglobin in said second bound fraction;
(e) passing a second beam of light through said mixture, said second beam having a wavelength maximum whereby it is absorbed by said product to give a second absorbance due to said HbAlc;
(f) measuring said second absorbance; and
(g) determining the percentage of HbAlc in said blood sample from the magnitude of said first and second abosrbances.
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Abstract
A method for determining glycosylated hemoglobin in a diluted blood sample includes binding of glycosylated hemoglobin to a specific monoclonal antibody and binding of nonglycosylated hemoglobin to a polyclonal antibody. The polyclonal antibody binds to all hemoglobin fractions and blocks the peroxidase activity thereof, except glycosylated hemoglobin bound to the monoclonal antibody which retains peroxidase activity. A beam of light having a wavelength of about 416 nm is passed through the assay medium and is absorbed by all hemoglobin fractions, bound or unbound, and this absorbance thereby provides a measure of total hemoglobin. Hydrogen peroxide and a peroxidase substrate are added. The substrate is oxidized by the peroxide to a product having an absorbance at a wavelength different from 416 nm, the oxidation being catalyzed by the retained peroxidase activity of the bound glycosylated hemoglobin. This absorbance provides a measure of glycosylated hemoglobin. From the two absorbances, the percentage of glycosylated hemoglobin in the total hemoglobin of the sample may be calculated. The invention includes a kit of materials useful in performing the assay method of the invention.
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Citations
18 Claims
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1. A method for determining the percentage of glycosylated hemoglobin fraction HbAlc in the total hemoglobin for a blood sample comprising:
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(a) preparing a mixture by contacting a blood smaple in a diluent with a monoclonal antibody specific for the glycoside portion of HbAlc and an antibody specific for the here portion of total hemoglobin whereby HbAlc in said sample binds to said monoclonal antibody to give a first bound fraction and nonglycosylated hemoglobin in said sample binds to said antibody to give a second bound fraction; (b) passing a first beam of light having a wavelength maximum of about 416 nm through said mixture, whereby said first and second bound fractions absorb said ligth to give a first absorbance due to said total hemoglobin; (c) measuring said first absorbance; (d) adding to said mixture a source of peroxide and a peroxidase substrate whereby peroxide from said source reacts with said substrate to give a product, said reaction being catalyzed by HbAlc in said first bound fraction but not by nonglycosylated hemoglobin in said second bound fraction; (e) passing a second beam of light through said mixture, said second beam having a wavelength maximum whereby it is absorbed by said product to give a second absorbance due to said HbAlc; (f) measuring said second absorbance; and (g) determining the percentage of HbAlc in said blood sample from the magnitude of said first and second abosrbances. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method for determining the percentage of glycosylated hemoglobin fraction HbAlc in the total hemoglobin of a blood sample comprising:
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(a) preparing a first mixture by contacting a first portion of a blood sample in a diluent with a source of peroxide and a first increment of a peroxidase substrate whereby said peroxide reacts with said substrate to give a first increment of a product, said reaction being catalyzed by the total hemoglobin in said sample; (b) passing a first beam of light through said first mixture, said first beam having a wavelength maximum whereby it is absorbed by said first increment of product to give a first absorbance due to said total hemoglobin in said sample; (c) measuring said first absorbance, (d) preparing a second mixture by contacting a second portion of said blood sample in said diluent with a first antibody specific for HbAlc and a second antibody specified for the here portion of total hemoglobin whereby HbAlc in said sample binds to said first antibody to give a bound fraction and nonglycosylated hemoglobin in said sample binds to said second antibody to give a second found fraction; (e) adding to said second mixture a source of peroxide and a second increment of said peroxidase substrate whereby said peroxide reacts with said second increment of substrate to give a second increment of said product, said reaction being catalyzed by said HbAlc in said first bound fraction but not by nonglycosylated hemoglobin in said second bound fraction; (f) passing a second beam of light through said second mixture, said second beam having a wavelength maximum whereby it is absorbed by said second increment of product to give a second absorbance due to said HbAlc in said sample; (g) measuring said second absorbance; and (h) determining the percentage of said HbAlc in said blood sample from the magnitude of said first and second absorbances. - View Dependent Claims (11, 12, 13)
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14. A method for determining the percentage of glycosylated hemoglobin fraction HbAlc in the total hemoglobin of a blood sample comprising:
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(a) preparing a mixture by contacting a blood smaple in phosphate buffered saline with a monclonal antibody specific for HbAlc and a polyclonal antibody specific for the heme portion of total hemoglobin whereby HbAlc in said sample binds to said monoclonal antibody to give a first bound faction and nonglycosylated hemoglobin in said sample binds to said polyclonal antibody to give a second bound fraction; (b) passing a first beam of light having a wavelength maximum of about 416 nm through said mixture, whereby said first and second bound fractions absorb said light to give a first absorbance due to said total hemoglobin; (c) measuring said first absorbance; (d) adding to said mixture hydrogen peroxide and leuco malachite green whereby said peroxide reacts with said leuco malachite green to give malachite green said reaction being catalyzed by HbAlc in said first bound fraction but not by nonglycosylated hemoglobin in said second bound fraction; (e) passing a second beam of light having a wavelength maximum of about 620 nm through said mixture, said second beam being absorbed by said malachite green to give a second absorbance due to said HbAlc; (f) measuring said second absorbance; and (g) determining the percentage of HbAlc in said blood sample from the magnitude of said first and second absorbances.
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- 15. A kit for determining the percentages of glycosylated hemoglobin fraction HbAlc in the total hemoglobin of a blood sample comprising a first antibody which specifically binds to HbAlc, a second antibody which specifically binds to nonglycosylated hemoglobin, a source of peroxide, a peroxidase substrate and one or more containers, said HbAlc, afte binding to said first antibody, catalyzing a reaction of said peroxide and said substrate to a product.
Specification