Source: http://www.google.com/patents/US7855079?dq=U.S.+Patent+%23+5,723,324
Timestamp: 2016-12-05 19:53:49
Document Index: 799968818

Matched Legal Cases: ['Application No. 04', 'Application No. 04', 'Application No. 04', 'Application No. 04', 'Application No. 04', 'Application No. 04', 'Application No. 04']

Patent US7855079 - Methods for assaying percentage of glycated hemoglobin - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inPatentsThe invention provides enzymatic methods for direct determination of percentage of glycated hemoglobin in blood samples without the need of a separated measurement of total hemoglobin content in blood samples. The methods utilizes one or two different types of oxidizing agents which selectively oxidize...http://www.google.com/patents/US7855079?utm_source=gb-gplus-sharePatent US7855079 - Methods for assaying percentage of glycated hemoglobinAdvanced Patent SearchTry the new Google Patents, with machine-classified Google Scholar results, and Japanese and South Korean patents.Publication numberUS7855079 B2Publication typeGrantApplication numberUS 11/881,179Publication dateDec 21, 2010Filing dateJul 25, 2007Priority dateJul 25, 2006Fee statusPaidAlso published asCA2654981A1, CA2654981C, CN101484809A, CN101484809B, EP2044444A1, EP2044444B1, US8318501, US20080096230, US20110189712, WO2008013874A1Publication number11881179, 881179, US 7855079 B2, US 7855079B2, US-B2-7855079, US7855079 B2, US7855079B2InventorsChong-Sheng Yuan, Abhijit Datta, Limin LiuOriginal AssigneeGeneral AtomicsExport CitationBiBTeX, EndNote, RefManPatent Citations (74), Non-Patent Citations (63), Referenced by (7), Classifications (17), Legal Events (2) External Links: USPTO, USPTO Assignment, EspacenetMethods for assaying percentage of glycated hemoglobin
US 7855079 B2Abstract
The invention provides enzymatic methods for direct determination of percentage of glycated hemoglobin in blood samples without the need of a separated measurement of total hemoglobin content in blood samples. The methods utilizes one or two different types of oxidizing agents which selectively oxidize low-molecular weight reducing substances and high-molecular weight (mainly hemoglobin) reducing substances in blood samples, coupled with enzymatic reactions catalyzed by proteases, fructosyl amino acid oxidase, and peroxidase. The invention provides kits for performing the methods of the invention.
1. A method for directly assaying percentage of glycated hemoglobin A1c in a blood sample without measuring the total hemoglobin in the blood sample in a separate process, said method comprising:
a) contacting protein fragments containing glycated peptides or glycated amino acids with a fructosyl amino acid oxidase to generate hydrogen peroxide (H2O2), wherein the protein fragments are generated by contacting the blood sample with 1) a lysing buffer which releases hemoglobin from red blood cells in the blood sample; 2) an oxidizing agent which is a tetrazolium salt, and 3) a protease which digests glycated hemoglobin into glycated peptides or glycated amino acids, wherein the protease is a neutral protease of Aspergillus or Bacillus origin and generates glycated glycine, glycated valine, glycated lysine residue, or a glycated peptide comprising glycated glycine, glycated valine or glycated lysine residue; wherein the fructosyl amino acid oxidase comprises the amino acid sequence set forth in SEQ ID NO:1;
b) contacting H2O2 generated in step a) with a color forming substance in the presence of a peroxidase to generate a measurable signal; and
c) determining percentage of glycated hemoglobin A1c by correlating the measured value in step b) to a percentage of glycated hemoglobin A1c in a calibration curve without measuring the total hemoglobin in the blood sample separately.
2. The method of claim 1, wherein the lysing buffer contains the oxidizing agent.
3. The method of claim 1 or 2, wherein the lysing buffer contains the protease.
4. The method of claim 1, wherein the oxidizing agent and the protease are formulated in a single composition.
5. The method of claim 1, wherein the fructosyl amino acid oxidase, the peroxidase, and the color forming substance are formulated in a single composition.
6. The method of claim 1, wherein tetrazolium salt is 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt or 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt.
7. The method of claim 1, wherein the color forming substance is N-Carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)-diphenylamine sodium salt (DA-64), N,N,N′N′,N″,N″-Hexa(3-sulfopropyl)-4,4′,4″,-triamino-triphenylmethane hexasodium salt (TPM-PS), or 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-phenothiazine sodium salt (DA-67).
8. The method of claim 1, wherein the blood sample is a whole blood.
9. The method of claim 1, wherein the protease generates a glycated peptide from about 2 to about 30 amino acid residues.
10. The method of claim 1, wherein the peroxidase is horseradish peroxidase.
11. The method of claim 1, wherein the protein fragments containing the glycated peptide or glycated amino acid are contacted with the fructosyl amino acid oxidase and the peroxidase sequentially or simultaneously.
12. The method of claim 1, further comprising using the percentage of glycated hemoglobin A1c for prognosing or diagnosing a disease or disorder.
13. A kit for directly assaying percentage of glycated hemoglobin A1c in a blood sample without the need of a separated measurement of total hemoglobin content in blood samples, said kit comprising:
a) a lysing buffer which lyses blood cells to release hemoglobin;
b) an oxidizing agent, wherein the oxidizing agent is a tetrazolium salt;
c) a protease which hydrolyzes hemoglobin into protein fragments containing glycated peptides or glycated amino acids, wherein the protease is a neutral protease of Aspergillus or Bacillus origin, wherein the protease generates glycated glycine, glycated valine, glycated lysine residue, or a glycated peptide comprising glycated glycine, glycated valine or glycated lysine residue;
d) a fructosyl amino acid oxidase which reacts with glycated peptides and glycated amino acids to generate hydrogen peroxide (H2O2), wherein the fructosyl amino acid oxidase comprises the amino acid sequence set forth in SEQ ID NO:1;
e) a peroxidase and a color forming substance;
f) calibrator(s) with known percentage of glycated hemoglobin A1c for use in constructing a calibration curve; and
g) instructions to perform the method of claim 1.
14. The kit of claim 13, wherein the oxidizing agent is contained in the lysing buffer.
15. The kit of claim 13 or 14, wherein the protease is contained in the lysing buffer.
16. The kit of claim 13, wherein the oxidizing agent and the protease are formulated in a single composition.
17. The kit of claim 13, wherein the fructosyl amino acid oxidase, the peroxidase, and the color forming substance are formulated in a single composition.
18. The kit of claim 13, wherein tetrazolium salt is 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt or 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt.
19. The kit of claim 13, wherein the color forming substance is N-Carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)-diphenylamine sodium salt (DA-64), N,N,N′N′,N″,N″-Hexa(3-sulfopropyl)-4,4′,4″,-triamino-triphenylmethane hexasodium salt (TPM-PS), or 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-phenothiazine sodium salt (DA-67).
20. The kit of claim 13, wherein the protease generates a glycated peptide from about 2 to about 30 amino acid residues.
21. The kit of claim 13, wherein the peroxidase is horseradish peroxidase. Description
This application claims the priority benefit of provisional patent applications U.S. Ser. Nos. 60/833,390, filed Jul. 25, 2006, and 60/858,809, filed Nov. 13, 2006, all of which are incorporated herein in their entirety by reference.
The invention provides direct enzymatic assay for determining percentage of glycated hemoglobin in a blood sample.
A glycated protein is a substance which is produced by the non-enzymatic and irreversible binding of the amino group of an amino acid constituting a protein, with the aldehyde group of a reducing sugar such as aldose. See e.g., U.S. Pat. No. 6,127,138. Such a non-enzymatic and irreversible binding reaction is also called “Amadori rearrangement,” and therefore the above-mentioned glycated protein may also be called “Amadori compound” in some cases.
Nonenzymatic glycation of proteins has been implicated in the development of certain diseases, e.g., diabetic complications and the aging process (Takahashi et al., J. Biol. Chem., 272 (19):12505-7 (1997); and Baynes and Monnier, Prog. Clin. Biol. Res., 304:1-410 (1989)). This reaction leads to dysfunction of target molecules through formation of sugar adducts and cross-links. Considerable interest has focused on the Amadori product that is the most important “early” modification during nonenzymatic glycation in vitro and in vivo.
Various assays for glycated proteins are known. For example, U.S. Pat. No. 6,127,138 discloses that a sample containing a glycated protein is treated with Protease XIV or a from Aspergillus genus, thereafter (or while treating the sample with the above protease) FAOD (fructosyl amino acid oxidase) is caused to react with the sample so as to measure the amount of oxygen consumed by the FAOD reaction or the amount of the resultant reaction product, thereby to measure the glycated protein.
In another example, U.S. Pat. No. 6,008,006 discloses that the amount of glycated proteins in a sample can be quantified by reacting the sample with first a reagent which is a combination of a protease and a peroxidase and second with a ketoamine oxidase. U.S. Pat. No. 6,008,006 also discloses a kit which contains the combined peroxidase/protease enzyme reagent and also the ketoamine oxidase. U.S. Pub. No. 2005/0014935 also discloses methods and kits for measuring amount of glycated protein using a chimeric amadoriase. U.S. Pub. No. 2003/0162242 and EP 1304385 A1 also disclose methods of selectively determining glycated hemoglobin.
Previously described methods for determining percentage of glycated hemoglobin A1c require a separate measurement of total hemoglobin in the samples. When a chemistry analyzer is used to determine the value of percentage of glycated hemoglobin A1c, a dual channel format is required. In this format, two separate assays are performed to determine 1) glycated hemoglobin A1c concentration, and 2) total hemoglobin concentration in the samples; and followed by calculating the ratio of glycated HbA1c to total hemoglobin to obtain percentage of HbA1c.
All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.
The invention provides methods for direct determination of percentage of glycated hemoglobin in a blood sample without the need of a separated measurement of total hemoglobin in the sample. Since there is no need for a separate measurement of total hemoglobin and no need for a ratio calculation step, the present methods can be fully automated and used with various chemical analyzers in a single channel format.
In one aspect, the present invention provides methods for directly assaying percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in a blood sample without measuring the total hemoglobin in the blood sample in a separate process, said method comprising: a) contacting protein fragments containing glycated peptides or glycated amino acids with a fructosyl amino acid oxidase to generate hydrogen peroxide (H2O2), wherein the protein fragments are generated by contacting the blood sample with 1) a lysing buffer which releases hemoglobin from red blood cells in the blood sample; 2) a first oxidizing agent which selectively oxidizes low molecular weight reducing substances; 3) a second oxidizing agent which selectively oxidizes high molecular weight reducing substances, and 4) a protease which digests glycated hemoglobin into glycated peptides or glycated amino acids; b) contacting H2O2 generated in step a) with a color forming substance in the presence of a peroxidase to generate a measurable signal; and c) determining percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in the sample by applying the signal generated in step b) to a calibration curve without measuring the total hemoglobin in the blood sample separately.
In some embodiments, the first oxidizing agent is Dess-Martin periodinane or N-ethylmaleimide, and wherein the second oxidizing agent is a tetrazolium salt.
In some embodiments, the lysing buffer contains the first oxidizing agent and/or the second oxidizing agent. In some embodiments, the lysing buffer contains the first oxidizing agent, the second oxidizing agent, and the protease. In some embodiments, the lysing buffer contains the protease.
In some embodiments, the first oxidizing agent and the second oxidizing agent are formulated in a single composition. In some embodiments, the first oxidizing agent and the second oxidizing agent are formulated in a separate composition. In some embodiments, the protease is formulated in a single composition with the first oxidizing agent or the second oxidizing agent. In some embodiments, the first oxidizing agent, the second oxidizing agent, and the protease are formulated in a single composition. In some embodiments, the fructosyl amino acid oxidase, the peroxidase, and the color forming substance are formulated in a single composition.
In another aspect, the invention provides methods for directly assaying percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in a blood sample without measuring the total hemoglobin in the blood sample in a separate process, said method comprising: a) contacting protein fragments containing glycated peptides or glycated amino acids with a fructosyl amino acid oxidase to generate hydrogen peroxide (H2O2), wherein the protein fragments are generated by contacting the blood sample with 1) a lysing buffer which releases hemoglobin from red blood cells in the blood sample; 2) an oxidizing agent which is a tetrazolium salt, and 3) a protease which digests glycated hemoglobin into glycated peptides or glycated amino acids; b) contacting H2O2 generated in step a) with a color forming substance in the presence of a peroxidase to generate a measurable signal; and c) determining percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in the sample by applying the signal generated in step b) to a calibration curve without measuring the total hemoglobin in the blood sample separately.
In some embodiments, the lysing buffer contains the oxidizing agent. In some embodiments, the lysing buffer contains the protease. In some embodiments, the oxidizing agent and the protease are formulated in a single composition.
In some embodiments, tetrazolium salt is 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt or 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt.
In some embodiments, the color forming substance is N-Carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)-diphenylamine sodium salt (DA-64), N,N,N′N′,N″,N″-Hexa(3-sulfopropyl)-4,4′,4″,-triamino-triphenylmethane hexasodium salt (TPM-PS), or 110-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-phenothiazine sodium salt (DA-67).
In some embodiments, the blood sample is a whole blood or collected blood cells.
In some embodiments, the protease is an endo-type protease or an exo-type protease. In some embodiments, the protease is selected from the group consisting of proteinase K, pronase E, ananine, thermolysin, subtilisin and cow pancreas proteases. In some embodiments, the protease is a neutral protease of Aspergillus or Bacillus origin. In some embodiments, the protease generates a glycated peptide from about 2 to about 30 amino acid residues. In some embodiments, the protease generates glycated glycine, glycated valine or glycated lysine residue or a glycated peptide comprising glycated glycine, glycated valine or glycated lysine residue.
In some embodiments, the peroxidase is horseradish peroxidase.
In some embodiments, the protein fragments containing the glycated peptide or glycated amino acid are contacted with the fructosyl amino acid oxidase and the peroxidase sequentially or simultaneously.
In some embodiments, the fructosyl amino acid oxidase comprises the amino acid sequence set forth in SEQ ID NO: 1
(MGGSGDDDDLALAVTKSSSLLIVGAGTWGTSTALHLARRGYTNVTVLDP
YPVPSAISAGNDVNKVISSGQYSNNKDEIEVNEILAEEAFNGWKNDPLFK
PYYHDTGLLMSACSQEGLDRLGVRVRPGEDPNLVELTRPEQFRKLAPEGV
LQGDFPGWKGYFARSGAGWAHARNALVAAAREAQRMGVKFVTGTPQGRVV
TLIFENNDVKGAVTGDGKIWRAERTFLCAGASAGQFLDFKNQLRPTAWTL
VHIALKPEERALYKNIPVIFNIERGFFFEPDEERGEIKICDEHPGYTNMV
QSADGTMMSIPFEKTQIPKEAETRVRALLKETMPQLADRPFSFARICWCA
DTANREFLIDRHPQYHSLVLGCGASGRGFKYLPSIGNLIVDAMEGKVPQK
IHELIKWNPDIAANRNWRDTLGRFGGPNRVMDFHDVKEWTNVQYRDISKL
KGELEGLPIPNPLLRTGHHHHHH).
In some embodiments, the method is used in the prognosis or diagnosis of a disease or disorder. In some embodiments, the disease or disorder is diabetes.
In another aspect, the invention provides kits for directly assaying percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in a blood sample without the need of a separate measurement of total hemoglobin content in blood samples, comprising: a) a lysing buffer that lyses blood cells to release hemoglobin; b) a first oxidizing agent that selectively oxidizes low molecular weight reducing substances; c) a second oxidizing agent that selectively oxidizes high molecular weight reducing substances; d) a protease that hydrolyzes hemoglobin into protein fragments containing glycated peptides or glycated amino acids; e) a fructosyl amino acid oxidase that reacts with glycated peptides and glycated amino acids to generate hydrogen peroxide (H2O2); f) a peroxidase and a color forming substance; and g) calibrator(s) with known percentage of glycated hemoglobin or known percentage of glycated hemoglobin A1c for use in constructing a calibration curve. The kit may further comprise instructions to perform the methods described herein.
In some embodiments, the first oxidizing agent and/or the second oxidizing agent are contained in the lysing buffer. In some embodiments, the first oxidizing agent and/or the second oxidizing agent are contained in the same buffer with the protease.
In another aspect, the invention provides kits for directly assaying percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in a blood sample without the need of a separated measurement of total hemoglobin content in blood samples, said kit comprising: a) a lysing buffer that lyses blood cells to release hemoglobin; b) an oxidizing agent, wherein the oxidizing agent is a tetrazolium salt; c) a protease that hydrolyzes hemoglobin into protein fragments containing glycated peptides or glycated amino acids; d) a fructosyl amino acid oxidase that reacts with glycated peptides and glycated amino acids to generate hydrogen peroxide (H2O2); e) a peroxidase and a color forming substance; and f) calibrator(s) with known percentage of glycated hemoglobin or known percentage of glycated hemoglobin A1c for use in constructing a calibration curve. The kit may further comprise instructions to perform the methods described herein.
In some embodiments, the oxidizing agent is contained in the lysing buffer. In some embodiments, the protease is contained in the lysing buffer. In some embodiments, the oxidizing agent and the protease are formulated in a single composition.
FIG. 1 shows the timeline of the single channel HbA1c enzymatic assay procedure.
FIG. 2 shows a calibration curve for enzymatic HbA1c assay. X-axis shows percentage of glycated hemoglobin A1c known for the calibration sample; and Y-axis shows the corresponding difference in absorbance value at 700 nm between 8 min and 5 min after adding reagent R1a and R1b.
FIG. 3 shows the correlation between the single channel enzymatic hemoglobin A1c assay described in Example 1 and Tosoh's HPLC method. Y axis shows the HbA1c value measured using the single channel enzymatic hemoglobin A1c assay described in Example 1 for samples; and X-axis shows HbA1c value measured using the Tosoh HPLC method for the corresponding samples.
FIG. 4 shows a calibration curve for enzymatic HbA1c assay using one oxidizing agent as described in Example 2. X-axis shows percentage of glycated hemoglobin A1c known for the calibration sample; and Y-axis shows the corresponding difference in absorbance value at 700 mm between 8 min and 5 min after adding reagent R1a and R1b.
The invention provides enzymatic methods for direct determination of percentage of glycated hemoglobin in blood samples without the need of a separate measurement of total hemoglobin content in blood samples. In one aspect, the methods utilizes two different types of oxidizing agents which selectively oxidize low-molecular weight reducing substances (mainly ascorbic acid and free thio-containing molecules) and high-molecular weight reducing substances (mainly hemoglobin) in blood samples, coupled with enzymatic reactions catalyzed by proteases, fructosyl amino acid oxidase, and peroxidase. In another aspect, the methods utilizes one type of oxidizing agent which selectively oxidize high-molecular weight reducing substances in blood samples, coupled with enzymatic reactions catalyzed by proteases, fructosyl amino acid oxidase, and peroxidase. The invention also provides kits for performing the methods of the invention.
As used herein, “a” or “an” means “at least one” or “one or more.”
As used herein, a “fructosyl amino acid oxidase” or (FAOD) refers to an enzyme catalyzing the oxidative deglycation of Amadori products to yield corresponding amino acids, glucosone, and H2O2, as shown in the following reaction:
R1—CO—CH2—NH—R2+O2+H2O→R1—CO—CHO+R2—NH2+H2O2 wherein R1 represents the aldose residue of a reducing sugar and R2 represents a residue of an amino acid, protein or peptide. Other synonyms of amadoriase include amadoriase, fructosyl amine:oxygen oxidoreductase (FAOO), and fructosyl valine oxidase (FVO). For purposes herein, the name “fructosyl amino acid oxidase” is used herein, although all such chemical synonyms are contemplated. “Fructosyl amino acid oxidase” also encompasses a functional fragment or a derivative that still substantially retain its enzymatic activity catalyzing the oxidative deglycation of Amadori products to yield corresponding amino acids, glucosone, and H2O2. Typically, a functional fragment or derivative retains at least 50% of its amadoriase activity. Preferably, a functional fragment or derivative retains at least 60%, 70%, 80%, 90%, 95%, 99% or 100% of its amadoriase activity. It is also intended that a fructosyl amino acid oxidase can include conservative amino acid substitutions that do not substantially alter its activity. Suitable conservative substitutions of amino acids are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson, et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. Co., p. 224). Such exemplary substitutions are preferably made in accordance with those set forth in TABLE 1 as follows:
Original residue
Conservative substitution
Gly; Ser
Gln; His
Gln (Q)
Ala; Pro
Asn; Gln
Leu; Val
Leu (L)
Ile; Val
Arg; Gln; Glu
Leu; Tyr; Ile
Met; Leu; Tyr
Trp (W)
Trp; Phe
Ile; Leu
Other substitutions are also permissible and may be determined empirically or in accord with known conservative substitutions.
As used herein, “peroxidase” refers to an enzyme that catalyses a host of reactions in which hydrogen peroxide is a specific oxidizing agent and a wide range of substrates act as electron donors. It is intended to encompass a peroxidase with conservative amino acid substitutions that do not substantially alter its activity. The chief commercially available peroxidase is horseradish peroxidase.
As used herein, a “composition” refers to any mixture of two or more products or compounds. It may be a solution, a suspension, a liquid, a powder, a paste, aqueous, non-aqueous, or any combination thereof.
B. Methods of Directly Assaying Percentage of Glycated Hemoglobin
In another aspect, the present invention is directed to a method for directly assaying percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c, said method comprising: a) lysing red blood cells in a blood sample with a lysing buffer to release hemoglobin; b) oxidizing the lysate with a first oxidizing agent which selectively oxidizes low molecular weight reducing substances; c) oxidizing the lysate with a second oxidizing agent which selectively oxidizes high molecular weight reducing substances; d) contacting the lysate with a protease to form protein fragments containing glycated peptides and/or glycated amino acids; e) contacting the protein fragments with a fructosyl amino acid oxidase to generate hydrogen peroxide (H2O2); f) allowing oxidization of a color forming substance by H2O2 generated in step e) in the presence of a peroxidase under Trinder reaction and by unreacted second oxidizing agent to generate a measurable signal; and g) assessing the signal generated in step f); and h) determining the percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in the sample by comparing the signal to a calibration curve without measuring the total hemoglobin in the blood sample separately.
In another aspect, the invention provides a method for directly assaying percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c without measuring total hemoglobin separately in a blood sample, said method comprising: a) lysing red blood cells in a blood sample with a lysing buffer to release hemoglobin; b) oxidizing the lysate with a first oxidizing agent, wherein the first oxidizing agent is Dess-Martin periodinane and/or N-ethylmaleimide; c) oxidizing the lysate with a second oxidizing agent, wherein the second oxidizing agent is a tetrazolium salt; d) contacting the lysate with a protease to form protein fragments containing glycated peptides and/or glycated amino acids; e) contacting the protein fragments with a fructosyl amino acid oxidase to generate hydrogen peroxide (H2O2); f) allowing oxidization of a color forming substance by H2O2 generated in step e) in the presence of a peroxidase under Trinder reaction to generate a measurable signal; g) assessing the signal generated in step f); and h) determining the percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in the sample by comparing the signal to a calibration curve without measuring the total hemoglobin in the blood sample separately.
The blood sample may be lysed, oxidized by the first oxidizing agent, oxidized by the second oxidizing agent, and the lysate fragmented by the protease simultaneously or by separate steps. Any combination of two or more of steps may be performed simultaneously. In some embodiments, steps involving lysing the red blood cells in the sample and oxidizing by the first oxidizing agent, lysing the red blood cells and oxidizing by the second oxidizing agent, oxidizing with the first oxidizing agent and the second oxidizing agent, or lysing the red blood cells and oxidizing with the first oxidizing agent and the second oxidizing agent are performed simultaneously. In some embodiments, steps involving lysing the red blood cells and fragmenting the lysate by the protease, oxidizing with the first oxidizing agent and fragmenting the lysate by the protease, oxidizing with the second oxidizing agent and fragmenting the lysate by the protease, or oxidizing with the first oxidizing agent and the second oxidizing agent and fragmenting the lysate by the protease are performed simultaneously. In some embodiments, the steps involving lysing the red blood cells, oxidizing with the first oxidizing agent and the second oxidizing agent, and fragmenting the lysate are performed simultaneously. In some embodiments, the first oxidizing agent and/or the second oxidizing agent are contained in the lysis buffer, or added into the blood sample at the same time that the lysis buffer is added. In some embodiments, the protease is also included in the lysis buffer with the first and the second oxidizing agent or added into the blood sample at the same time that the lysis buffer and the first and the second oxidizing agents are added into the blood sample. In some embodiments, the first and/or the second oxidizing agent are in the same solution with the protease solution before adding into the red blood cell lysate. In some embodiments, the first oxidizing agent and lysing buffer are formulated in a single composition. In some embodiments, the protease and the first oxidizing agent or the second oxidizing agent are formulated in a single composition. In some embodiments, the first and the second oxidizing agents are formulated in a single composition. In some embodiments, the first and the second oxidizing agents are formulated in a separate composition. In some embodiments, the lysis buffer contains the protease, or the protease is added into the blood sample at the same time that the lysis buffer is added.
The first oxidizing agent is a type of oxidizing agent that selectively oxidizes low molecular weight (M.W.<3000) reducing substances. The first oxidizing agent has higher oxidizing power toward low molecular weight reducing substances than high molecular weight (M.W.>3000) reducing substances. Examples of low molecular weight substances in the blood sample are ascorbic acid and free thio containing molecules. Examples of first oxidizing agent are Dess-Martin periodinane and N-ethyl maleimide. Other examples of first oxidizing agents are sodium iodoacetate, sodium periodate, and Chloramine-T. In some embodiments, more than one first oxidizing agents (e.g., both Dess-Martin periodinane and N-ethyl maleimide) are used.
The second oxidizing agent is a type of oxidizing agent that selectively oxidizes high molecular weight (M.W.>3000) reducing substances. The second oxidizing agent has higher oxidizing power toward high molecular weight reducing substances than low molecular weight reducing substance. An example of high molecular weight substances in the blood sample is hemoglobin. An example of second oxidizing agent is a tetrazolium salt (e.g., 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt, or 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt). Other examples of second oxidizing agent are sodium dodecyl sulfate, potassium ferricyanide (III), and potassium iodate. In some embodiments, more than one second oxidizing agent is used.
In another aspect, the invention provides methods for directly assaying percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in a blood sample without measuring the total hemoglobin in the blood sample in a separate process, said method comprising: a) contacting protein fragments containing glycated peptides or glycated amino acids with a fructosyl amino acid oxidase to generate hydrogen peroxide (H2O2), wherein the protein fragments are generated by contacting the blood sample with 1) a lysing buffer which releases hemoglobin from red blood cells in the blood sample; 2) an oxidizing agent which is a tetrazolium salt, and 3) a protease which digests glycated hemoglobin into glycated peptides or glycated amino acids; b) contacting H2O2 generated in step a) with a color forming substance in the presence of a peroxidase to generate a measurable signal; and c) determining percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in the sample by applying the signal generated in step b) to a calibration curve without measuring the total hemoglobin in the blood sample separately. Any tetrazolium salt described herein may be used. Steps a) and b) may be performed sequentially or simultaneously. In some embodiments, the lysing buffer contains the oxidizing agent. In some embodiments, the lysing buffer contains the protease. In some embodiments, the lysing buffer contains the oxidizing agent and the protease. In some embodiments, the oxidizing agent and the protease are formulated in a single composition.
Blood samples that can be assayed using the present methods include whole blood or collected blood cells. The red blood cells in the blood sample are lysed in a lysing buffer to release hemoglobin. Any lysing buffer (e.g., in the acidic or alkaline pH ranges) that can lyse the red blood cells and release the hemoglobin can be used. Lysing buffer generally contains a detergent, such as Triton (e.g., Triton X-100), Tween (e.g., Tween 20), sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), tetradecyltrimethylammonium bromide (TTAB), polyoxyethylene lauryl ethers (POEs), and Nonidet P-40 (NP-40).
Any suitable protease can be used in the present methods. Either an endo-type protease or an exo-type protease can be used. Exemplary endo-type proteases include trypsin, α-chymotrypsin, subtilisin, proteinase K, papain, cathepsin B, pepsin, thermolysin, protease XVII, protease XXI, lysyl-endopeptidase, prolether and bromelain F. Exemplary exo-type proteases include an aminopeptidase or a carboxypeptidase. In one example, the protease is proteinase K, pronase E, ananine, thermolysin, subtilisin or cow pancreas proteases. Metaloproteases and neutral proteinases from Aspergillus sps, Alicyclobacillus sps, and Bacillus sps may also be used.
The protease can be used to generate a glycated peptide of any suitable size. For example, the protease can be used to generate a glycated peptide from about 2 to about 30 amino acid residues. In another example, the protease is used to generate glycated glycine, glycated valine or glycated lysine residue or a glycated peptide comprising glycated glycine, glycated valine or glycated lysine residue.
Glycated peptide and/or glycated amino acid are contacted with a fructosyl amino acid oxidase. Any fructosyl amino acid oxidase (FAOD) can be used. Fructosyl amino acid oxidase may be purified or recombinantly produced. Any naturally occurring species may be used. In one example, the FAOD used is of Aspergillus sp. origin (See, e.g., Takahashi et al., J. Biol. Chem. 272(6):3437-43, 1997). Other fructosyl amino acid oxidase, e.g., disclosed in GenBank Accession No. U82830 (Takahashi et al., J. Biol. Chem., 272(19):12505-12507 (1997) and disclosed U.S. Pat. No. 6,127,138 can also be used. A functional fragment or a derivative of an amadoriase that still substantially retain its enzymatic activity catalyzing the oxidative deglycation of Amadori products to yield corresponding amino acids, glucosone, and H2O2 can also be used.
Normally, a functional fragment or a derivative of an amadoriase retains at least 50% of its enzymatic activity. Preferably, a functional fragment or a derivative of an amadoriase retain at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of its enzymatic activity.
Any of the chimeric proteins having the enzymatic activities of FAOD described in the U.S. Pub. No. 2005/0014935 can be used. In some embodiments, the fructosyl amino acid oxidase comprises from the N-terminus to C-terminus: a) a first peptidyl fragment comprising a bacterial leader sequence from about 5 to about 30 amino acid residues; and b) a second peptidyl fragment comprising an FAOD. In some embodiments, the FAOD comprises the following amino acid sequence:
MGGSGDDDDLALAVTKSSSLLIVGAGTWGTSTALHLARRGYTNVTVLDPY
PVPSAISAGNDVNKVISSGQYSNNKDEIEVNEILAEEAFNGWKNDPLFKP
YYHDTGLLMSACSQEGLDRLGVRVRPGEDPNLVELTRPEQFRKLAPEGVL
QGDFPGWKGYFARSGAGWAHARNALVAAAREAQRMGVKFVTGTPQGRVVT
LIFENNDVKGAVTGDGKIWRAERTFLCAGASAGQFLDFKNQLRPTAWTLV
HIALKPEERALYKNIPVIFNIERGFFFEPDEERGEIKICDEHPGYTNMVQ
SADGTMMSIPFEKTQIPKEAETRVRALLKETMPQLADRPFSFARICWCAD
TANREFLIDRHPQYHSLVLGCGASGRGFKYLPSIGNLIVDAMEGKVPQKI
HELIKWNPDIAANRNWRDTLGRFGGPNRVMDFHDVKEWTNVQYRDISKLK
GELEGLPIPNPLLRTGHHHHHH.
The chimeric protein may be produced in bacterial cells, such as E. coli. The protein produced may be purified and assayed for the enzymatic activities. Assays for enzymatic activities of FAOD are known in the art (See e.g., Takahashi et al., J. Biol. Chem., 272(6):3437-43 (1997) and U.S. Pat. No. 6,127,138). Four exemplary assays for enzymatic activities of amadoriases are disclosed in Takahashi et al., J. Biol. Chem., 272(6):3437-43 (1997).
The hydrogen peroxide generated in the reaction catalyzed by the fructosyl amino acid oxidase is assessed by Trinder reaction. A color forming substance and a peroxidase is added into the reaction, the color forming substance is oxidized by the hydrogen peroxide to form a color substance such as quinoneimine or Bindschedler's green derivatives and H2O. The amount of quinoneimine or Bindschedler's green product generated can be determined by measuring absorbance between about 500 nm to about 800 nm (such as around 700 nm). Without wishing to be bound by theory, the second oxidizing agent unreacted with the high molecular weight substance in the blood lysate may also react with the color forming substance to the form colored product, and thus the absorbance measured reflects the percentage of total glycated hemoglobin and percentage of glycated hemoglobin A1c in the blood sample. Examples of color forming substances are N-(Carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)-diphenylamine sodium salt (DA-64), N,N,N′,N′,N″,N″-Hexa(3-sulfopropyl)-4,4′,4″,-triamino-triphenylmethane hexasodium salt (TPM-PS), and 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-phenothiazine sodium salt (DA-67). An example of peroxidase is a horseradish peroxidase.
In some embodiments, the glycated peptide and/or glycated amino acid are contacted with the fructosyl amino acid oxidase and the peroxidase sequentially or simultaneously. In some embodiments, the FAOD, the peroxidase, and the color forming substance are formulated in a single composition. In some embodiments, the FAOD, the peroxidase, and the color forming substance are formulated in a different composition and are added into the reaction at the same time or at different times.
The percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in the blood sample is determined by comparing the absorbance (e.g., absorbance at around 700 nm) to a calibration curve. Using the present methods, the percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in the blood sample is determined without measuring the total hemoglobin in the blood sample separately. The calibration curve is established using calibrator, i.e., samples (including blood samples and artificial calibrators) with known percentage of glycated hemoglobin or known percentage of glycated hemoglobin A1c. See, e.g., Example 1.
In some embodiments, the calibration curve is prepared by determining signal levels (e.g., absorbance at around 700 nm) for calibration samples by performing the same steps as the unknown samples without measuring the total hemoglobin separately; and graphing the correlation between the signal levels of the calibration samples and the known percentage of glycated hemoglobin or known percentage of glycated hemoglobin A1c of the calibration samples. For example, whole blood samples having percentages of glycated hemoglobin A1c value assigned by comparison to a suitable higher order reference material can be used as calibrators. Alternative, the percentage of glycated hemoglobin A1c may be determined by another recognized method such as HPLC. Calibrators are tested the same way as the unknown samples using the methods described herein. The absorbance values measured for calibrators are plotted against the expected HbA1c value to establish the calibration curve.
Calibrators other than whole blood sample may also be used to establish a calibration curve. Hemolysate samples (lysed blood samples), glycated peptides, glycated amino acid, and glycated amino acid derivatives in a suitable buffered protein matrix solution having percentage of glycated hemoglobin A1c value assigned by comparison to a suitable higher order reference material can be used as artificial calibrators. For example, calibration samples may be prepared in a phosphate buffered solution with 10% BSA and appropriate amounts of synthesized fructosyl propylamine (glycated amino acid) corresponding to various percentage of HbA1c (e.g., from 5% o 12%). Artificial calibrators are tested the same way as unknown samples except that the lysing step may not be used. The absorbance values measured for these calibrators are plotted against the expected HbA1c value to establish the calibration curve. Artificial calibrators may be lyophilized or stabilized for extended shelf life.
The present methods can be used for any suitable purpose. Preferably, the method used in the prognosis or diagnosis of a disease or disorder, e.g., diabetes.
C. Kits for Assaying Percentage of Glycated Hemoglobin
The invention also provides a kit for assaying percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c without measuring the total hemoglobin separately in a blood sample, said kit comprising: a) a lysing buffer that lyses blood cells to release hemoglobin; b) a first oxidizing agent that selectively oxidizes low molecular weight reducing substances; c) a second oxidizing agent that selectively oxidizes high molecular weight reducing substances; d) a protease that hydrolyzes hemoglobin into protein fragments containing glycated peptides or glycated amino acids; e) a fructosyl amino acid oxidase that reacts with glycated peptides and glycated amino acids to generate hydrogen peroxide (H2O2); f) a peroxidase and a color forming substance; and g) glycated hemoglobin or glycated hemoglobin A1c calibrator(s) (i.e., calibrator(s) with known percentage of glycated hemoglobin or known percentage of glycated hemoglobin A1c) for use in constructing a calibration curve.
In some embodiments, the first oxidizing agent Dess-Martin periodinane and/or N-ethylmaleimide. In some embodiments, the second oxidizing agent is a tetrazolium salt.
In some embodiments, the lysing buffer contain the first oxidizing agent and/or the second oxidizing agent. In some embodiments, the lysing buffer contains the protease. In some embodiments, the lysing buffer contains the first oxidizing agent, the second oxidizing agent, and the protease. In some embodiments, the first oxidizing agent and the second oxidizing agent are formulated in a single composition. In some embodiments, the first oxidizing agent and the second oxidizing agent are formulated in a separate composition. In some embodiments, the protease is formulated in a single composition with the first oxidizing agent and/or the second oxidizing agent.
The invention also provides a kit for directly assaying percentage of total glycated hemoglobin or percentage of glycated hemoglobin A1c in a blood sample without the need of a separated measurement of total hemoglobin content in blood samples, said kit comprising: a) a lysing buffer that lyses blood cells to release hemoglobin; b) an oxidizing agent, wherein the oxidizing agent is a tetrazolium salt; c) a protease that hydrolyzes hemoglobin into protein fragments containing glycated peptides or glycated amino acids; d) a fructosyl amino acid oxidase that reacts with glycated peptides and glycated amino acids to generate hydrogen peroxide (H2O2); e) a peroxidase and a color forming substance; and f) calibrator(s) with known percentage of glycated hemoglobin or known percentage of glycated hemoglobin A1c for use in constructing a calibration curve. In some embodiments, the oxidizing agent is contained in the lysing buffer. In some embodiments, the protease is contained in the lysing buffer. In some embodiments, the oxidizing agent and the protease are contained in the lysing buffer. In some embodiments, the protease and oxidizing agent are formulated in a single composition.
In some embodiments, the fructosyl amino acid oxidase and the peroxidase are formulated in a single composition. In some embodiments, the calibrator is a blood sample with known percentage of glycated hemoglobin A1c, which may be in lyophilized form or in solution.
In some embodiments, the kit comprises a lysing buffer, a R1a reagent, a R1b reagent, and a R2 reagent. In some embodiments, the lysing buffer comprises a first oxidizing agent (e.g., N-ethylmaleimide and/or Dess Martin Periodinane). In some embodiments, the R1a reagent comprises a protease and a first oxidizing agent (e.g., N-ethylmaleimide and/or Dess Martin Periodinane). In some embodiments, the R1b reagent comprises a first oxidizing agent (e.g., N-ethylmaleimide and/or Dess Martin Periodinane) and a second oxidizing agent (e.g., a tetrazolium salt). In some embodiments, the R2 reagent comprises a fructosyl amino acid oxidase, a peroxidase (e.g., horseradish peroxidase), and a color forming substance (e.g., DA-64). For examples, the lysing buffer may contain 0.1-10% Triton X-100 (e.g., about 0.1%, about 0.2%, about 0.5%, about 1%, about 2.5%, about 5%, about 7.5%, about 10%); 5-100 mM CHES (e.g., about 5 mM, about 10 mM, about 25 mM, about 50 mM, about 75 mM, about 100 mM), pH about 8.7; 0.1-50 mM N-ethylmaleimide (e.g., about 0.1 mM, about 0.5 mM, about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM); 0.1-5% SDS (e.g., about 0.15%, about 0.25%, about 0.35%, about 0.45%, about 0.55%, about 0.75%, about 1%, about 2.5%, about 5%); 0.001-1 KU/ml catalase (e.g., about 1 U/ml, about 2 U/ml, about 3 U/ml, about 4 U/ml, about 5 U/ml, about 10 U/ml, about 50 U/ml, about 100 U/ml, about 500 U/ml, about 1 KU/ml); 0.001-1 KU/ml ascorbate oxidase (e.g., about 1 U/ml, about 2 U/ml, about 4 U/ml, about 5 U/ml, about 10 U/ml, about 50 U/ml, about 100 U/ml, about 1 KU/ml). The R1a reagent may contain 0.1-10 KU/ml Bacillus sp Protease (e.g., about 0.1 KU/ml, about 2 KU/ml, about 3.0 KU/ml, about 3.5 KU/ml, about 4.0 KU/ml, about 4.5 KU/ml, about 5 KU/ml, about 10 KU/ml); 1-100 mM MES (e.g., about 1 mM, about 5 mM, about 10 mM, about 25 mM, about 50 mM, about 100 mM), pH about 7.0; 1-10 mM CaCl2 (e.g., about 1 mM, about 2.5 mM, about 5 mM, about 7.5 mM, about 10 mM); 0.01-10 mM Dess Martin Periodinane (e.g., about 0.01 mM, about 0.015 mM, about 0.02 mM, about 0.05 mM, about 0.1 mM, about 5 mM, about 10 mM); 0.01-5 mg/ml methyl 4-hydroxybenzoate sodium salt (e.g., about 0.01 mg/ml, about 0.05 mg/ml, about 0.1 mg/ml, about 1 mg/ml, about 5 mg/ml); and 0.001-1 mg/ml geneticin (G418) (e.g., about 0.001 mg/ml, about 0.01 mg/ml, about 0.1 mg/ml, about 1 mg/ml). The R1b reagent may contain 0.1-50 mM MES hydrate (e.g., about 0.1 mM, about 0.5 mM, about 1.0 mM, about 10 mM, about 25 mM, about 50 mM); 0.1-50 mM WST-3 (2-(4-Iodopenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) (e.g., about 0.1 mM, about 0.5 mM, about 1 mM, about 2.5 mM, about 2.6 mM, about 2.7 mM, about 2.8 mM, about 2.9 mM, about 3.0 mM, about 5 mM, about 10 mM, about 25 mM, about 50 mM); and 0.01-10 mM Dess Martin Periodinane (e.g., about 0.01 mM, about 0.04 mM, about 0.05 mM, about 0.06 mM, about 0.07 mM, about 0.08 mM, about 0.09 mM, about 0.1 mM, about 1 mM, about 5 mM, about 10 mM). The R2 reagent may contain 0.01-10 KU/ml fructosyl valine oxidase (e.g., about 0.01 KU/ml, about 0.012 KU/ml, about 0.013 KU/ml, about 0.0135 KU/ml, about 0.014 KU/ml, about 0.0145 KU/ml, about 0.015 KU/ml, about 0.0155 KU/ml, about 0.016 KU/ml, about 0.05 KU/ml, about 0.1 KU/ml, about 1 KU/ml, about 5 KU/ml, about 10 KU/ml); 1-50 mM Tris-HCl (e.g., about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 50 mM), pH about 8.0; 0.1-10% Triton X-100 (e.g., about 0.1%, about 0.2%, about 0.5%, about 1%, about 2.5%, about 5%, about 7.5%, about 10%); 0.01-10 KU/ml horse radish peroxidase (HRP) (e.g., about 0.01 KU/ml, about 0.05 KU/ml, about 0.08 KU/ml, about 0.09 KU/ml, about 0.1 KU/ml, about 1.0 KU/ml, about 5 KU/ml, about 10 KU/ml); 0.01-10 mM DA-64 (e.g., about 0.01 mM, about 0.05 mM, about 0.075 mM, about 0.08 mM, about 0.085 mM, about 0.09 mM, about 0.1 mM, about 1 mM, about 5 mM, about 10 mM); and 0.01-10 mg/ml geneticin (G418) (e.g., about 0.01 mg/ml, 0.05 mg/ml, about 0.1 mg/ml, about 5 mg/ml, about 10 mg/ml). The kit may further comprise instructions to practice methods described herein. The kit may be used in described in detail in Example 1.
The kits of the invention may be in any suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Kits may further comprise instructions for practicing any of the methods described herein.
Single Channel HbA1c Enzymatic Assay
This single channel HbA1c test is an enzymatic assay in which samples are lysed and reacted with agents to eliminate low molecular weight and high molecular weight signal interfering substances. The lysed whole blood samples are subjected to extensive protease digestion with Bacillus sp protease. This process releases amino acids including glycated valine from the hemoglobin beta chains. Glycated valine is then served as a substrate for specific recombinant fructosyl valine oxidase (FVO) enzyme, produced in E. coli. The recombinant FVO specifically can cleave N-terminal valine and produce hydrogen peroxide in the presence of selective agents. This, in turn, is measured using a horse radish peroxidase (POD) catalyzed reaction and a suitable chromagen. The HbA1c concentration is expressed directly as % HbA1c by use of a suitable calibration curve.
1. Reagent Compositions.
Lysis buffer: 50 mM CHES pH 9.4, 2% Triton X-100, 3 mM Dess-Martin Periodinane, and 2.5 mM N-ethyl Maleimide.
Reagent R1a: 25 mM MES buffer pH 6.5, 5 mM CaCl2, 1000 U/ml neutral protease (Toyobo Co., Ltd.), 2 mM N-ethyl Maleimide.
Reagent R1b: 25 mM MES pH 6.5, 150 mM sodium chloride, 5 mM Dess-Martin Periodinane, 2 mM WST3 (2-(4-Iodopenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (manufactured by Dojindo Laboratories)).
Reagent R2: 25 mM Tris, pH 8.2, 5 U/ml fructosyl amino acid oxidase having the amino acid sequence shown in SEQ ID NO: 1, 50 U/ml Horse Radish Peroxidase, and 0.5 mM chromagen (N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino) diphenylamine sodium salt (product name DA-64, manufactured by Wako).
II. Assay Procedure
Lysis buffer (500 μL) was dispensed in a suitable container such as a sample cup or an eppendorf microfuge tube. Prior to testing, whole blood samples were mixed by gentle inversion to resuspend settled erythrocytes. Fully resuspended whole blood sample (40) was mixed gently with the lysis buffer using a suitable pipettor without creating foam. The mixture was then incubated for 5 to 10 minutes at room temperature. Complete lysis was observed when the mixture became a clear red solution without any particulate matter.
Reagents R1a and R1b were mixed in 70:30 volume ratio prior to use. Reagent R1b was poured into R1a, and the reagents were mixed gently by inversion to form Reagent R1ab.
Reagent R1ab (170 μL) and lysate (20 μL) were added into a cuvette, and mixed. The cuvette was incubated at 37° C. for 5 minutes. The reaction can also be carried out at room temperature. After the incubation, 50 μL of Reagent R2 was added into the cuvette. The absorbance at 700 nm was monitored for 3 minutes. The absorbance value was calculated for calibrators, controls and samples by subtracting O.D. value at A2 (absorbance at 3 min after the addition of R2) from O.D. value at A1 (absorbance right after the addition of R2), i.e., ΔA700=(A2−A1). The timeline of the reaction is shown in FIG. 1. Values of unknown samples were determined by use of a calibration curve shown, e.g., in FIG. 2 which was represented directly in HbA1c % units.
The calibration curve (FIG. 2) was prepared using data from measuring ΔA700 for three standard samples with known percentage of glycated hemoglobin A1c (6.25%, 10.00%, and 15.00%) following the procedure described above. The calibrators were prepared by testing suitable whole blood material for HbA1c values using HPLC method. The whole blood material used for calibration could be lyophilized or stabilized for extended shelf life.
As shown in Table 2, the value of percentage of glycated hemoglobin A1c obtained using the method described above (column under “obtained value”) correlates with expected value in the sample. The expected value for a sample was obtained from HPLC. The ranges for the expected value indicate acceptable value ranges.
TABLE 2 HbA1c (%) Samples Expected Value (HPLC) Obtained Value 1 6.2 (5.27-7.13) 6.84 2 12.2 (10.37-14.03) 12.13 3 5.9 (5.0-6.8) 5.15 4 11.1 (9.4-12.7) 11.18 5 5.4 5.21 6 9.1 9.30 7 5.4 5.71 8 9.0 9.17 III. Precision of the Single Channel Enzymatic Hemoglobin A1c Assay
The within-run precision was evaluated with two different % HbA1c level fresh whole blood samples (sample ID 10810285 low HbA1c and sample ID 10810244 high HbA1c) replicated 16 times. The evaluation was done using the Hitachi 917 auto-analyzer instrument and the single channel enzymatic hemoglobin A1c assay described in this example. Normal Control and pathonormal controls were included in the study.
The whole blood samples with verified % HbA1c values used for this study were obtained from a certified commercial source, ProMedDx, LLC (10 Commerce Way, Norton, Mass. 2766) and came with an IRB certification that protocols, informed consent, used to collect samples were IRB approved.
Table 3 below shows the precision of the single channel enzymatic hemoglobin A1c assay.
TABLE 3 ID 10810285 ID 10810244 (% HbA1c) (% HbA1c) Mean value 4.8% 8.2% Intra run SD 0.07 0.05 Intra run CV % 1.4% 0.6% IV. Accuracy of the Single Channel Enzymatic Hemoglobin A1c Assay
To demonstrate accuracy, the single channel enzymatic hemoglobin A1c assay was used with individual whole blood samples (ID series depicted below) and compared to Tosoh's HbA1c HPLC assay, which is the currently marketed HbA1c device (Tosoh G7: HbA1c Variant Analysis Mode). The accuracy study tests were performed on the Hitachi 917 Auto-analyzer instrument.
The whole blood samples with verified HbA1c values used for this study were from a certified commercial source, ProMedDx, LLC and came with an IRB certification that protocols, informed consent, used to collect samples were IRB approved.
The comparison study included 30 test samples and the results obtained are shown in Table 4 below. “Tosoh % HbA1c” indicates values obtained using Tosoh's HbA1c HPLC method for the samples. “DZ % HbA1c” indicates corresponding values obtained using the single channel enzymatic hemoglobin A1c assay described in this example.
Fresh Whole Blood Sample ID
Tosoh % HbA1c
DZ % HbA1c
10810257
10897226
10897227
10897229
10897230
10845039
10845043
10845044
10845045
10845059
10810281
10897261
10897272
10897278
10897231
10897234
10897238
10897239
10897241
10845060
10845063
10845065
10845066
10845068
10897285
10897286
10897290
10897295
DZ Ctl L1 Lot CON100405B-1
DZ Ctl L2 Lot CON200405B-1
FIG. 3 shows the HbA1c value (%) obtained using the single channel enzymatic hemoglobin A1c assay described in this example plotted against results obtained with Tosoh's HPLC methods. As shown in FIG. 3, the slope was 1.05; the correlation coefficient between the two methods was 0.96; and the y intercept was −0.367.
Single Channel HbA1c Enzymatic Assay Using One Oxidizing Agent
The procedures of single channel HbA1c test in this example were similar to the procedures described in Example 1 except only one oxidizing agent was used for oxidizing reducing substances in the blood samples.
I. Reagent Compositions
Lysis buffer: 50 mM CHES pH 9.4, and 2% Triton X-100.
Reagent R1a: 25 mM MES buffer pH 6.5, 5 mM CaCl2, 1000 U/ml neutral protease (Toyobo Co., Ltd.).
Reagent R1b: 25 mM MES pH 6.5, 150 mM sodium chloride, and 2 mM WST3 (2-(4-Iodopenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (manufactured by Dojindo Laboratories)).
Lysis buffer (500 μL) was dispensed in a suitable container such as a sample cup or an eppendorf microfuge tube. Prior to testing, whole blood samples were mixed by gentle inversion to resuspend settled erythrocytes. Fully resuspended whole blood sample (40 μL) was mixed gently with the lysis buffer using a suitable pipettor without creating foam. The mixture was then incubated for 5 to 10 minutes at room temperature. Complete lysis was observed when the mixture became a clear red solution without any particulate matter.
Reagent R1ab (170 μL) and lysate (20 μL) were added into a cuvette, and mixed. The cuvette was incubated at 37° C. for 5 minutes. After the incubation, 50 μL of Reagent R2 was added into the cuvette. The absorbance at 700 nm was monitored for 3 minutes. The absorbance value was calculated for calibrators, controls and samples by subtracting O.D. value at A2 (absorbance at 3 min after the addition of R2) from O.D. value at A1 (absorbance right after the addition of R2), i.e., ΔA700=(A2−A1). The timeline of the reaction is shown in FIG. 1.
FIG. 4 shows the data from measuring ΔA700 (Y-axis) for the samples following the procedure described above plotted against known percentage of glycated hemoglobin A1c. FIG. 4 indicates that percentage of glycated hemoglobin A1c can also be determined directly without a separate measurement of total hemoglobin using a reagent system with a single oxidizing agent tetrazolium, though the results (accuracy and correlation) were not as good as those obtained with a reagent system with two oxidizing agents as described in Example 1.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, descriptions and examples should not be construed as limiting the scope of the invention, which is delineated by the appended claims.
Patent CitationsCited PatentFiling datePublication dateApplicantTitleUS2713581Feb 23, 1949Jul 19, 1955Ellis Foster CoCertain tetrazolium salts and process for preparing themUS4588684Apr 26, 1983May 13, 1986Chiron Corporationa-Factor and its processing signalsUS4837331Nov 13, 1986Jun 6, 1989Wako-Pure Chemical Industries, Ltd.Stabilization of tetrazolium salts with cyclodextrinsUS4847195Nov 24, 1987Jul 11, 1989Syntex (U.S.A.) Inc.Glucose-6-phosphate dehydrogenase conjugates useful in polyiodothyronine assaysUS4948729Mar 17, 1988Aug 14, 1990Cetus CorporationProduction of soluble recombinant proteinsUS5013647Apr 3, 1987May 7, 1991Boehringer Mannheim GmbhRemoval of interfering reducing substances with tetrazolium salts in redox measurement reactionsUS5030563Jan 26, 1990Jul 9, 1991Genetics Institute, Inc.Bacterial hypersecretion using mutant repressor sequenceUS5120420Mar 31, 1989Jun 9, 1992Matsushita Electric Industrial Co., Ltd.Biosensor and a process for preparation thereofUS5137821Dec 13, 1989Aug 11, 1992Toyo Jozo Company, Ltd.Gene and process of making glucose-6-phosphate dehydrogenaseUS5171670May 12, 1989Dec 15, 1992The General Hospital CorporationRecombinant dna method for production of parathyroid hormoneUS5196314Feb 26, 1991Mar 23, 1993Boehringer Mannheim GmbhProcess and reagent for the determination of substrates or enzyme activitiesUS5229286Jul 30, 1991Jul 20, 1993Boehringer Mannheim GmbhCloning and overexpression of glucose-6-phosphate dehydrogenase from leuconostoc dextranicusUS5244796Oct 12, 1990Sep 14, 1993Syracuse UniversityCloned leuconostoc mesenteroides glucose-6-phosphate dehydrogenase genes and method of making glucose-6-phospate dehydrogenaseUS5308770Feb 25, 1993May 3, 1994Boehringer Mannheim GmbhCloning and overexpression of glucose-6-phosphate dehydrogenase from Leuconostoc dextranicusUS5312759Dec 20, 1989May 17, 1994Iatron Laboratories, Inc.Method for measurement of fructosamines using 1,2-quinonesUS5320732Jun 11, 1993Jun 14, 1994Matsushita Electric Industrial Co., Ltd.Biosensor and measuring apparatus using the sameUS5344770Oct 26, 1993Sep 6, 1994Kao CorporationBacillus SP. ferm BP-3376 and a method for its use to produce an alkaline proteinase K-16US5681529Aug 23, 1995Oct 28, 1997Nihon Medi-Physics Co., Ltd.Biological fluid analyzing deviceUS5682884Jul 27, 1994Nov 4, 1997Medisense, Inc.Strip electrode with screen printingUS5710248Jul 29, 1996Jan 20, 1998University Of Iowa Research FoundationPeptide tag for immunodetection and immunopurificationUS5712138Sep 29, 1995Jan 27, 1998Kyoto Daiichi Kagaku Co., Ltd.Fructosyl amino acid oxidaseUS5789221Mar 1, 1995Aug 4, 1998Kyogo Daiichi Kagaku Co., Ltd.Fructosyl amino acid oxidase and process for producing the sameUS5824527Apr 11, 1996Oct 20, 1998Kyoto Daiichi Kagaku Co., Ltd.Fructosyl amino acid oxidase and process for producing the sameUS5856104Mar 7, 1997Jan 5, 1999Affymetrix, Inc.Polymorphisms in the glucose-6 phosphate dehydrogenase locusUS5879921Nov 7, 1996Mar 9, 1999Novo Nordisk A/SRecombinant expression of a glucose oxidase from a cladosporium strainUS5885811Jun 5, 1995Mar 23, 1999University Of MarylandLeader sequence inducing a post-translational modification of polypeptides in bacteria gene therefor and subtilin variant of enhanced stability and activityUS5914250Dec 8, 1997Jun 22, 1999University Of MarylandLeader sequence inducing a post-translational modification of polypeptides in bacteria, gene therefor, and subtilin variant of enhanced stability and activityUS5948659Feb 26, 1998Sep 7, 1999Kyoto Daiichi Kagaku Co., Ltd.Recombinant fructosyl amino acid oxidaseUS5948665Feb 11, 1997Sep 7, 1999Oriental Yeast Co. Ltd.Hexokinase obtained from thermophilic yeast Kluyveromyces fragilisUS5972294Mar 16, 1998Oct 26, 1999Lifescan, Inc.Reagent test strip for determination of blood glucoseUS5972671Feb 25, 1998Oct 26, 1999Kyoto Daiichi Kagaku Co., Ltd.Fructosyl amino acid oxidase and process for producing the sameUS5985591Nov 19, 1998Nov 16, 1999Kyoto Daiichi Kagaku Co., Ltd.Method for the determination of glycosylated proteinsUS6008006May 3, 1996Dec 28, 1999Genzyme Limited Et Al.Determination of glycated proteinsUS6069297Dec 5, 1997May 30, 2000Sloan-Kettering Institute For Cancer ResearchGlucose-6-phosphate dehydrogenase deficient mice and methods of using sameUS6127138Apr 24, 1998Oct 3, 2000Kyoto Daiichi Kagaku Co., Ltd.Method of enzymatically measuring glycated proteinUS6127345Nov 3, 1997Oct 3, 2000Smithkline Beecham CorporationPolynucleotides encoding the glucose 6-phosphate dehydrogenase of Streptococcus pneumoniaeUS6194200Dec 18, 1992Feb 27, 2001OncogenExpression systems for preparation of polypeptides in prokaryotic cellsUS6329161Sep 22, 2000Dec 11, 2001Therasense, Inc.Subcutaneous glucose electrodeUS6352835Nov 15, 1999Mar 5, 2002Kyoto Daiichi Kagaku Co. Ltd.Method of measuring substance in sample using a redox reactionUS6451574Sep 23, 1997Sep 17, 2002The Procter & Gamble CompanyProteinase K variants having decreased adsorption and increased hydrolysisUS6514720Sep 5, 2001Feb 4, 2003Kyoto Daiichi Kagaku Co., Ltd.Method of measuring substance in sample using a redox reactionUS6863800Feb 1, 2002Mar 8, 2005Abbott LaboratoriesElectrochemical biosensor strip for analysis of liquid samplesUS7153666 *Jul 17, 2003Dec 26, 2006General AtomicsMethods and compositions for determination of glycated proteinsUS7235378 *Jul 12, 2001Jun 26, 2007Arkray, Inc.Method of selectively determining glycated hemoglobinUS20020009779May 21, 2001Jan 24, 2002Meyers Rachel A.50365, a novel hexokinase family member and uses thereforUS20030162242Jul 12, 2001Aug 28, 2003Satoshi YoneharaMethod of selectively determining glycated hemoglobinUS20040063213 *Sep 27, 2001Apr 1, 2004Kaoru HiraiAssay method with the use of redox reactionUS20050014935Jul 17, 2003Jan 20, 2005Chong-Sheng YuanMethods and compositions for determination of glycated proteinsUS20080299597 *May 13, 2008Dec 4, 2008Chong-Sheng YuanMethods for assaying percentage of glycated hemoglobinEP0121352A1Mar 6, 1984Oct 10, 1984The Public Health Laboratory Service BoardA leader sequence to promote the secretion of gene productsEP0186643A1Nov 19, 1985Jul 2, 1986Région Wallonne représentée par l'Exécutif Régional WallonMethod for the production of vector plasmids capable of transforming an Escherichia coli host bacterium and to promote and control the expression of heterologous DNAEP0196864A2Mar 25, 1986Oct 8, 1986Cetus CorporationAlkaline phosphatase-mediated processing and secretion of recombinant proteins, DNA sequences for use therein and cells transformed using such sequencesEP0239931A2Mar 26, 1987Oct 7, 1987Boehringer Mannheim GmbhProcess and reagent for the determination of substrates or enzyme activitiesEP0821064A2Jul 19, 1997Jan 28, 1998Kyoto Daiichi Kagaku Co., Ltd.Recombinant fructosyl amino acid oxidaseEP1223224A1Sep 29, 2000Jul 17, 2002Arai, Ayumi, Kikkoman CorporationMethod of assaying glycoproteinEP1304385A1Jul 12, 2001Apr 23, 2003ARKRAY, Inc.Method of selectively determining glycated hemoglobinGB738585A Title not availableGB1513488A Title not availableWO1989003886A1Oct 28, 1988May 5, 1989Oncogen, A Limited PartnershipExpression systems for preparation of polypeptides in prokaryotic cellsWO1990012113A1Apr 12, 1990Oct 18, 1990Enzymatics, Inc.The use of fluid insoluble oxidizing agents to eliminate interfering substances in oxidation-reduction measuring systemsWO1993006125A1Sep 21, 1992Apr 1, 1993The United States Of America, Represented By The Secretary, Department Of Health And Human ServicesISOLATION AND CHARACTERIZATION OF cDNA OF PLASMODIUM FALCIPARUM GLUCOSE-6-PHOSPHATE DEHYDROGENASEWO1996028556A2Mar 6, 1996Sep 19, 1996The Procter & Gamble CompanyProteinase k variants having decreased adsorption and increased hydrolysisWO2000028041A1Nov 5, 1999May 18, 2000Microbiological Research AuthorityDelivery of superoxide dismutase to neuronal cellsWO2001047968A1Dec 18, 2000Jul 5, 2001Fudan UniversityA novel polypeptide-hexokinase protein 12 and polynucleotide encoding said polypeptideWO2001068694A1Feb 26, 2001Sep 20, 2001Shanghai Biowindow Gene Development Inc.A novel polypeptide - human hexokinase protein 10 and a polynucleotide sequence encoding the sameWO2001090325A2May 21, 2001Nov 29, 2001Millennium Pharmaceuticals, Inc.50365, a hexokinase family member and uses thereofWO2001090378A1May 14, 2001Nov 29, 2001Shanghai Biowindow Gene Development Inc.Novel polypeptide--- a human hexokinase protein and polynucleotide encoding itWO2001098472A1Jun 15, 2001Dec 27, 2001Kyowa Hakko Kogyo Co., LtdNovel glucose-6-phosphate dehydrogenaseWO2002020795A1Jul 2, 2001Mar 14, 2002Biowindow Gene Development Inc. ShanghaiA novel polypeptide- hexokinase protein 9.68 and the polynucleotide encoding said polypeptideWO2002064760A2Feb 5, 2002Aug 22, 2002Roche Diagnostics GmbhExpression of recombinant proteinase k from tritirachium album in yeastWO2002072634A2Feb 8, 2002Sep 19, 2002Roche Diagnostics GmbhRecombinant proteinase kWO2003042389A1Nov 11, 2002May 22, 2003Basf AktiengesellschaftGenes coding for glucose-6-phosphate-dehydrogenase proteinsWO2005017136A1Jul 16, 2004Feb 24, 2005General AtomicsMethods and compositions for determination of glycated proteinsWO2008013874A1Jul 25, 2007Jan 31, 2008General AtomicsMethods for assaying percentage of glycated hemoglobin* Cited by examinerNon-Patent CitationsReference1Allgrove et al. (1988). Archives of Disease in Childhood 63:418-422.2Amendment After Final Action (37 C.F.R. Section 1.116) from U.S. Appl. No. 10/622,893, filed Jun. 22, 2006.3Amendment in Response to Non-Final Office Action from U.S. Appl. No. 10/622,893, filed Nov. 22, 2005.4Armbuster. (1987). Fructosamine: Clin. Chem. 33(12):2153-63.5Baker et al. (1991). Clin. Chem. 37(4):552-556.6Bastin et al. (1996). Mol. Biochem. Parasitology 77:235-239.7Baynes et al. eds. (1989). Prog. Clin. Biol. Res. 304:1-410.8Chen et al. (1998). BioTechniques 25(1):22-24.9Communication pursuant to Article 96(2) EPC, from EP Application No. 04 778 420.2-1222, dated Aug. 29, 2006.10Communication under Rule 51(4) EPC, from EP Application No. 04 778 420.2-1222, dated May 31, 2007.11Communication under Rule 51(4) EPC, from EP Application No. 04 778 420.2—1222, dated May 31, 2007.12Diazyme Laboratories Division, General Atomics, FAOX-TE Fact Sheet, 5 pages. (2004).13Entry into the European phase, from EP Application No. 04 778 420.2-1222, filed Feb. 17, 2006.14Entry into the European phase, from EP Application No. 04 778 420.2—1222, filed Feb. 17, 2006.15Final Office Action from U.S. Appl. No. 10/622,893, mailed on Feb. 23, 2006.16Fossati et al. (1980). Clin. Chem. 26(2):227-231.17Frederick et al. (1990). J. Biol. Chem. 265(7):3793-802.18Gerhardinger et al. (1995). The Journal of Biological Chemistry 270(1):218-224.19Hardy et al. (1989). J. Cell. Sci. Suppl. 11:29-43.20Hobom et al. (1995). Dev. Biol. Stand. 11:255-262.21International Preliminary Report on Patentability for PCT/US2004/022908, issued on Jan. 23, 2006, 6 pages.22International Search Report mailed on Jan. 26, 2005, for PCT Patent Application No. PCT/US2004/022908, 5 pages.23International Search Report mailed on Nov. 20, 2007, for PCT Application No. PCT/US2007/016774, filed Jul. 25, 2007, 2 pages.24International Search Report mailed on Nov. 20, 2007, for PCT Application No. PCT/US2007/016774, filed Jul. 25, 2007, five pages.25Invitrogen Catalogue. (2002). "pBAD TOPO TA Expression Kit," p. 75.26Kouzuma et al. (2002). Clin. Chimi, Acta. 324:61-71.27Kuhn et al. (1979). Mol. Gen. Genet. 167(3):235-241.28Luo, Y-C. et al. (2006, e-pub Sep. 5, 2006). "An Amperometric Uric Acid Biosensor Based on Modified Ir-C Electrode," Biosensors and Bioelectronics 22:482-488.29Michiels et al. (2001). Trends Microbiol. 9(4):164-168.30Nagelkerken et al. (1997). Electrophoresis 18:2694-98.31Non-Final Office Action from U.S. Appl. No. 10/622,893, mailed on Jul. 22, 2005.32Notice of Allowance and Examiner's Amendment from U.S. Appl. No. 10/622,893, mailed on Aug. 10, 2006.33Olah et al. (1994). Biochem. 221:94-102.34Peakman et al. (1992). Nucleic Acids Res. 20(22):6111-12.35Phillipou et al. (1988). Clin. Chem. 34(8):1561-64.36Prickett et al. (1989). BioTechniques 796):580-584.37Response to Office Action, from EP Application No. 04 778 420.2-1222, filed Mar. 7, 2007.38Response to Office Action, from EP Application No. 04 778 420.2—1222, filed Mar. 7, 2007.39Response to Restriction Requirement from U.S. Appl. No. 10/622,893, filed May 16, 2005.40Restriction Requirement from U.S. Appl. No. 10/622,893, mailed on Apr. 14, 2005.41Roesser et al. (1991). Nucleic Acids Res. 19(4):795-800.42Rudiger et al. (1997). BioTechniques 23(1):96-97.43Safer et al. (1988). FASEB J. 2(3):199-208.44Sakai et al. (1995). Biosci. Biotech. Biochem. 59(3):487-491.45Sakurabayashi et al. (2003). "New Enzymatic Assay for Glycohemoglobin," Clinical Chemistry 49(2):269-274.46 *Sakurabayashi et al., New Enzymatic Assay for Glycohemoglobin, Clinical Chemistry 49 (2): 26-274 (2003).47Schleicher et al. (1988). Clin. Chem. 34(2):320-323.48Sigma-Aldrich Catalog No. 82452, printed in Mar. 2, 2004, 1 page.49Takahashi et al. (1997). J. Biol. Chem. 272(19):12505-7.50Takahashi et al. (1997). J. Biol. Chem. 272(6):3437-43.51Tolbert et al. (1998). J. Neurochem. 70:113-119.52Tseng et al. (1996). Gene 169:287-288.53U.S. Appl. No. 11/595,209, filed Nov. 8, 2006, by Yuan et al.54Unknown. (1972). Biochemistry 11(9):1726-32.55Wang et al. (1996). Gene 169(1):53-58.56Wang, J. (2008, e-pub Dec. 23, 2007). "Electrochemical Glucose Biosensors," Chem. Rev. 108(2):814-825.57Watson et al. (1987). Molecular Biology of the Gene, 4th Edition. The Bejacmin/Cummings Pub. Co., p. 224.58Worthington Catalog, Proteinase K, located at , printed on Mar. 2, 2004, 4 pages.59Worthington Catalog, Proteinase K, located at <http://worthington-biochem.com/PROK.html>, printed on Mar. 2, 2004, 4 pages.60Written Opinion mailed on Nov. 20, 2007, for PCT Application No. PCT/US2007/016774, filed Jul. 25, 2007, 6 pages.61Xie et al. (1998). Endocrinology 139(11):4563-67.62Yoshida et al. (1995). Applied and Environmental Microbiology 61(12):4487-4489.63Yoshida et al. (1996). European Journal of Biochemistry 242(3):499-505.* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS8318501Nov 11, 2010Nov 27, 2012General AtomicsMethods for assaying percentage of glycated hemoglobinUS8338184 *Apr 8, 2011Dec 25, 2012General AtomicsMethods for assaying percentage of glycated hemoglobinUS8557591 *Nov 9, 2012Oct 15, 2013General AtomicsMethods for assaying percentage of glycated hemoglobinUS8673646May 13, 2009Mar 18, 2014General AtomicsElectrochemical biosensor for direct determination of percentage of glycated hemoglobinUS20100025264 *May 13, 2009Feb 4, 2010Chong-Sheng YuanElectrochemical biosensor for direct determination of percentage of glycated hemoglobinUS20110189712 *Nov 11, 2010Aug 4, 2011General AtomicsMethods for assaying percentage of glycated hemoglobinUS20110250627 *Apr 8, 2011Oct 13, 2011General AtomicsMethods for assaying percentage of glycated hemoglobin* Cited by examinerClassifications U.S. Classification436/67, 436/177, 435/25, 435/14, 436/66, 436/17, 436/522, 435/381, 435/2International ClassificationG01N33/72, G01N33/00Cooperative ClassificationY10T436/107497, G01N33/723, Y10T436/105831, Y10T436/25375, Y10T436/104998European ClassificationG01N33/72B2Legal EventsDateCodeEventDescriptionJan 14, 2008ASAssignmentOwner name: GENERAL ATOMICS, CALIFORNIAFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:YUAN, CHONG-SHENG;DATTA, ABHIJIT;LIU, LIMIN;REEL/FRAME:020361/0743;SIGNING DATES FROM 20071012 TO 20071018Owner name: GENERAL ATOMICS, CALIFORNIAFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:YUAN, CHONG-SHENG;DATTA, ABHIJIT;LIU, LIMIN;SIGNING DATES FROM 20071012 TO 20071018;REEL/FRAME:020361/0743May 28, 2014FPAYFee paymentYear of fee payment: 4RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services