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-10202632-B2
Publication Date
2019-02-12
Expiration Date
Abstract
A method for determining the amount of glycated hemoglobin (HbA1c), in which—if required—the erythrocytes in a sample are hemolyzed, the haemoglobin that is then released—if required—is contacted with a proteolytic agent and the glycated hemoglobin 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, the provision of the requisite proteolytic agent in the form of an inactivated protease is proposed, which is then only reactivated in situ. For the stabilization of the hemoglobin, which is unfolded at a very low pH in the range from 1 to 3, at least one suitable stabilizer should be present in the hemolysis solution, and, 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.
Core Innovation
The invention provides a method of determining the amount of glycated haemoglobin (HbA1c) in a sample by haemolysing erythrocytes to release the HbA1c contained therein. The released glycated haemoglobin 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 by the proteolytic agent.
The proteolytic agent is produced by bringing the released HbA1c into contact with at least two different solutions. One solution has a pH value in the range of 1 to 8 and contains a metalloprotease, a chelator for divalent metal ions, and Ca2+ and/or Mg2+, and the other solution contains 100 to 5000 μmol/l of a divalent metal ion selected from Fe2+, Mn2+, Co2+ and Zn2+.
The described approach includes stabilisation features for the assay chemistry. Protease stability is improved by combining chelator with Ca2+/Mg2+ in one solution while the divalent metal ions for reactivation are provided in a separate solution, haemoglobin stability is supported under low-pH hemolysis using phosphatidylcholine and/or 2-(methacryloyloxyethyl)-2'-(trimethylammoniumethyl)phosphate polymer/copolymer, and leuco dye inherent colouring and signal loss are reduced by phosphorus compounds of formula (I) and/or thio compounds, together with an SH group-trapping agent.
Claims Coverage
The partial content provides two independent claims: a method claim and a reagent kit claim. Across these, the claims center on a multi-solution protease production scheme with defined pH, chelator, divalent metal ion selection and ratios, and include dependent refinements for haemolysis formulation, degradation-product quantification chemistry, leuco dye stabilisation, and SH group trapping.
Multi-solution metalloprotease production with chelator, Ca2+/Mg2+ and reactivating divalent metal ions
A method of determining the amount of glycated haemoglobin (HbA1c) where haemolysis releases HbA1c and a proteolytic agent produces glycated haemoglobin degradation products, wherein the proteolytic agent is produced by contacting the released HbA1c with at least two different solutions: one solution having pH 1 to 8 and containing a metalloprotease, chelator for divalent metal ions at 0.1 to 2 mmol/l per 1000 kU/I of metalloprotease, and 0.5 to 10 mmol/l Ca2+ or 0.5 to 10 mmol/l Mg2+ with chelator:Ca2+ or chelator:Mg2+ molar ratio 1:2 to 1:20, and another solution containing 100 to 5000 μmol/l of a divalent metal ion selected from Fe2+, Mn2+, Co2+ and Zn2+.
Reagent kit with separate solutions for metalloprotease activation using chelator, Ca2+/Mg2+ and divalent metal ions
A reagent kit for use in a method determining the amount of glycated haemoglobin (HbA1c) comprising at least two different solutions in separate containers: one solution having pH 1 to 8 and containing a metalloprotease and per 1000 kU/I metalloprotease 0.1 to 2 mmol/l chelator for divalent metal ions, and 0.5 to 10 mmol/l Ca2+ or 0.5 to 10 mmol/l Mg2+, with chelator:Ca2+ or chelator:Mg2+ molar ratio 1:2 to 1:20, and the other solution containing 100 to 5000 μmol/l of a divalent metal ion selected from Fe2+, Mn2+, Co2+ and Zn2+.
The claim coverage is grounded in producing the proteolytic agent using at least two solutions with a defined pH 1 to 8 metalloprotease/chelator/Ca2+ or Mg2+ composition and a separate divalent metal reactivation solution using Fe2+, Mn2+, Co2+ or Zn2+, and in providing this arrangement as a reagent kit with separately contained solutions. Dependent claim refinements in the provided material further specify haemolysis-solution additives and pH 1 to 3, quantification chemistry using oxidases/peroxidase/leuco dye with a phosphorus compound of formula (I), optional thio compounds for leuco dye stabilisation, and SH group trapping using an SH group-trapping agent.
Stated Advantages
Improved protease stability across storage temperatures/times.
Improved haemoglobin stability under low-pH hemolysis (pH 1 to 3).
Reduced leuco dye inherent colouring and signal loss with selected stabilizers, including phosphorus stabilizers (formula (I)) and thio compounds, alone and in combination.
Documented Applications
Determining the amount of glycated haemoglobin (HbA1c) in a sample, using an enzymatic assay workflow that quantifies glycated haemoglobin degradation products.
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