Patent US4806468A successfully refreshed from Google Patents.
US4806468A
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Measurement of glycosylated hemoglobin by immunoassay
Assignee: Becton Dickinson and Co
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Filing: 1987-02-05
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Granted: 1989-02-21
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Expires: 2007-02-05
Abstract
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.
Inventors
- 👤 Daniel B. Wagner
- 👤 Ashoke Sinha
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 10:18
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/72; G01N33/721; G01N33/723; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S436/00; Y10S436/811; Y10T; Y10T436/00; Y10T436/25; Y10T436/25125
Claims Analysis
Total Claims: 22
Claim 1
Independent Claim
234 words
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.
Claim 2
Independent Claim
234 words
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.
Claim 3
Dependent Claim
32 words
The method in accordance with claim 1 wherein said antibody specific for the heme portion of total hemoglobin is selected from the group consisting of a monoclonal antibody and a polyclonal antibody.
Claim 4
Dependent Claim
23 words
The method in accordance with claim 1 wherein said monoclonal antibody and said antibody are added sequentially with intervening and following incubation periods.
Claim 5
Dependent Claim
26 words
The method in accordance with claim 1 wherein said monoclonal antibody and said antibody are added simultaneously to said blood sample followed by an incubation period.
Claim 6
Dependent Claim
28 words
The method in accordance with claim 1 wherein said source of peroxide is selected from the group consisting of hydrogen peroxide, an organic peroxide and a peroxide precursor.
Claim 7
Dependent Claim
30 words
The method in accordance with claim 5 wherein said peroxide precursor is selected from the group consisting of metaperborate, an inorganic peroxide and a mixture of glucose and glucose oxidase.
Claim 8
Dependent Claim
27 words
The method in accordance with claim 1 wherein said substrate is selected from the group consisting of 2,7-diaminofluorene, leuco malachite green, 3,5-diaminobenzidine and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt.
Claim 9
Dependent Claim
20 words
The method in accordance with claim 1 further comprising adding a lysing agent to said mixture subsequent to said contacting.
Claim 10
Dependent Claim
14 words
The method in accordance with claim 8 wherein said lysing agent is a surfactant.
Claim 11
Independent Claim
314 words
A method for determining the percentage of glycosylated hemoglobin fraction HbAlc in the total hemoglobin of a blood sample comprising: (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.
Claim 12
Independent Claim
314 words
A method for determining the percentage of glycosylated hemoglobin fraction HbAlc in the total hemoglobin of a blood sample comprising: (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.
Claim 13
Dependent Claim
13 words
The method in accordance with claim 10 wherein said source is hydrogen peroxide.
Claim 14
Dependent Claim
15 words
The method in accordance with claim 10 wherein said source is a hydrogen peroxide precursor.
Claim 15
Dependent Claim
19 words
The method in accordance with claim 10 wherein said first antibody is monoclonal and said second antibody is polyclonal.
Claim 16
Independent Claim
234 words
A method for determining the percentage of glycosylated hemoglobin fraction HbAlc in the total hemoglobin of a blood sample comprising: (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.
Claim 17
Independent Claim
234 words
A method for determining the percentage of glycosylated hemoglobin fraction HbAlc in the total hemoglobin of a blood sample comprising: (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.
Claim 18
Dependent Claim
69 words
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.
Claim 19
Dependent Claim
69 words
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.
Claim 20
Dependent Claim
19 words
The kit in accordance with claim 15 wherein said first antibody is monoclonal and said second antibody is polyclonal.
Claim 21
Dependent Claim
27 words
The kit in accordance with claim 15 wherein said source is selected from the group consisting of hydrogen peroxide, an organic peroxide and a hydrogen peroxide precursor.
Claim 22
Dependent Claim
35 words
The kit in accordance with claim 15 further comprising a solution useful in performing an assay method of the invention selected from the group consisting of saline, buffer, diluent and solution of an assay component.
References Cited (Backward Citations)
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