Enzymatic determination of HBA1c
Inventors
GORKA, Günther • Watazu, Yoshifumi • Metzmann, Erwin • LEIN, Alexandra • Müller, Holger • GRIMMLER, Matthias
Assignees
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Publication Number
US-10093959-B2
Publication Date
2018-10-09
Expiration Date
Abstract
A method for determining the amount of glycated haemoglobin (HbA1c), in which—if required—the erythrocytes in a sample are haemolysed, the haemoglobin that is then released—if required—is contacted with a proteolytic agent and the glycated haemoglobin degradation products obtained in this way or otherwise are quantified is disclosed. In order to provide such a process and reagents employable therein that has/have the property of sufficient stability of the chemical compounds that are essential to the reaction, for the stabilization of the haemoglobin which is unfolded at a very low pH in the range from 1 to 3, at least one suitable stabilizer is present in the haemolysis solution. Where a leuco dye is used in connection with the determination of the amount of HbA1c, it is proposed that the latter be stabilized with particular phosphine compounds and/or thio compounds, and, in particular embodiments, the requisite proteolytic agent is to be provided in the form of an inactivated protease which is then only reactivated in situ.
Core Innovation
The invention relates to determining the amount of glycated haemoglobin (HbA1c) in a sample by haemolysing erythrocytes to release HbA1c, producing glycated haemoglobin degradation products with a proteolytic agent, and determining the amount of HbA1c by quantifying the glycated haemoglobin degradation products produced. The haemolysis is effected by adding a haemolysis solution (H) having a pH-value in the range of 1 to 3, wherein the haemolysis solution contains a haemolytic detergent and at least one stabiliser selected from phosphatidylcholine, a 2-(methacryloxyethyl)-2'-(trimethyl ammoniumethyl)phosphate polymer, and/or a zwitterionic detergent.
The invention addresses reagent stability by stabilising proteolysis and protein/dye components. A proteolytic agent is stabilised during storage in an inactive or chelated state and is reactivated using specific divalent metal ions including Fe2+, Mn2+, Co2+ and Zn2+, together with chelator:Ca2+ and chelator:Mg2+ molar ratios within defined ranges. Haemoglobin is stabilised in the presence of haemolytic conditions at very low haemolysis solution pH values by using haemolytic detergents and at least one stabiliser selected from phosphatidylcholine, the specified phosphate-containing methacrylate polymer/copolymer, and/or zwitterionic detergents.
The invention further improves signal stability by stabilising the leuco dye used for quantification. The oxidation reaction produces hydrogen peroxide (H2O2), which is quantified via a peroxidase-driven colour reaction of a stabilised leuco dye. Stabilisation of the leuco dye includes phosphorus-containing stabilisers of a formula (IV) and/or thio compounds, and the leuco dye stabilisation approach includes the use of thio compounds selected from thiodiglycol, thiomalic acid, thionicontinomide, and thio-NAD, including mixtures thereof.
Claims Coverage
Two independent claims define the core coverage. Across them, the inventive features address low-pH haemolysis using specific detergent and stabiliser systems, proteolytic degradation and enzymatic oxidation-to-H2O2 quantification, and reagent kit compositions with stabilised protease and stabilised leuco dye systems using defined chemical classes and ranges.
Low-pH haemolysis with detergent and specified stabilisers
A method for determining HbA1c wherein haemolysis of erythrocytes is performed by adding a haemolysis solution (H) having a pH-value in the range of 1 to 3 and containing a haemolytic detergent and at least one stabiliser selected from phosphatidylcholine, 2-(methacryloxyethyl)-2’-(trimethyl ammoniumethyl)phosphate polymer, and a zwitterionic detergent, and mixtures thereof.
Proteolytic production of glycated haemoglobin degradation products and quantification
A method in which the released HbA1c is brought into contact with a proteolytic agent to produce glycated haemoglobin degradation products, and the amount of HbA1c is determined by quantification of the glycated haemoglobin degradation products produced.
Enzymatic oxidation to hydrogen peroxide with peroxidase and stabilised leuco dye readout
A method in which glycated haemoglobin degradation products are quantified by oxidation with fructosyl peptide oxidase or fructosyl amino acid oxidase to produce hydrogen peroxide (H2O2), and the hydrogen peroxide amount is determined via a peroxidase-driven colour reaction of a stabilised leuco dye using a phosphorus-containing stabiliser represented by formula (IV), optionally with thio compounds for dye stabilisation.
Reagent kit with separate solutions including low-pH haemolysis and enzymatic components
A reagent kit for determining HbA1c comprising separate containers with a haemolysis solution (H) of pH 1 to 3 containing a haemolytically acting detergent and 100 to 5000 μmol/L of a divalent metal ion selected from Fe2+, Mn2+, Co2+ and Zn2+, a first reagent solution (R1) containing fructosyl peptide oxidase or fructosyl amino acid oxidase and peroxidase, and a second reagent solution (R2) containing a protease, a chelator for divalent metal ions, Ca2+ or Mg2+ with chelator:Ca2+ and chelator:Mg2+ molar ratios in the range of 1:2 to 1:20, and a leuco dye; wherein the haemolysis solution includes the specified polymer and/or a phosphatidylcholine and/or a zwitterionic detergent.
Overall, the independent claims cover HbA1c determination combining low-pH haemolysis using a defined detergent system with specified stabilisers, proteolytic generation of glycated haemoglobin degradation products, enzymatic oxidation producing hydrogen peroxide with peroxidase and a stabilised leuco dye colour reaction, and reagent kit compositions in separated solutions that include defined divalent metal ion ranges, chelator/Ca2+/Mg2+ molar ratios, and leuco dye stabilisation components.
Stated Advantages
Improved reagent stability through stabilising the proteolytic agent and components used for quantification.
Reduced signal loss (described as improved stability of measured HbA1c signal over storage).
Stabilisation of the leuco dye used in the oxidation/peroxidase colour reaction.
Documented Applications
Enzymatic determination of the amount of glycated haemoglobin (HbA1c) in a sample.
Use of the described reagent kit in a method of determining HbA1c in a sample using separate solutions for haemolysis and enzymatic reactions.
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