Enzymatic HbA1c Determination Stabilizing Proteases and Leuco Dyes
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for determining glycated haemoglobin (HbA1c) face challenges in maintaining the stability of chemical and diagnostic reagents, especially during temperature fluctuations, which affects the accuracy and reproducibility of results.
Innovation Solution
A method involving haemolysis of erythrocytes to release HbA1c, followed by contact with a proteolytically acting agent to produce degradation products, with specific stabilisation of proteases and leuco dyes using pH-controlled solutions and chelators to maintain enzyme activity and prevent self-digestion, and the use of stabilisers like phosphatidylcholine to unfold and stabilise haemoglobin.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If proteases are used to produce glycated haemoglobin degradation products, then HbA1c determination can be performed, but the proteases undergo self-digestion and lose stability during storage
Solution Approach 1:
The patent applies preliminary anti-action by adding protease inhibitors to the reagent formulation before storage. These inhibitors prevent self-digestion of the protease during storage, maintaining enzyme stability. During the actual assay, the inhibitors are removed or inactivated, allowing the protease to function normally in producing glycated haemoglobin degradation products.
Solution Approach 2:
The patent uses protease inhibitors as intermediary substances that temporarily bind to the protease during storage to prevent self-digestion. These inhibitors act as mediators between the protease and the storage environment, protecting the enzyme without interfering with its intended function during the assay when the inhibitors are removed.
2Measurement precision
If leuco dyes are used for photometric measurement of hydrogen peroxide, then measurement sensitivity is improved, but autooxidation of the leuco dye causes non-specific blank value signals and increased spectral background
Solution Approach 1:
The patent extracts or removes the problematic autooxidation background signal by using selective filtration or computational methods to subtract the blank value caused by leuco dye autooxidation. This allows the specific signal from hydrogen peroxide reaction to be measured more accurately against a corrected baseline.
Solution Approach 2:
The patent converts the harmful autooxidation background signal into a beneficial reference by measuring the blank value in parallel and using it for calibration or subtraction. The autooxidation, while producing noise, provides a consistent baseline that can be mathematically removed to enhance the accuracy of the specific HbA1c measurement.
3Productivity
If haemoglobin is unfolded to improve protease accessibility, then degradation product production is enhanced, but haemoglobin denaturation and precipitation occur
Solution Approach 1:
The patent applies parameter changes by carefully controlling pH, temperature, and ionic strength to achieve partial unfolding of haemoglobin that enhances protease accessibility without causing complete denaturation and precipitation. By optimizing these parameters, the patent maintains haemoglobin in a state that is sufficiently unfolded for enzyme action while retaining solubility.
Solution Approach 2:
The patent uses partial action by inducing only the necessary degree of unfolding required for protease access, rather than complete denaturation. This controlled partial unfolding achieves the productivity goal of enhanced degradation product production while avoiding the harmful effect of excessive unfolding that leads to precipitation.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method ensures the stability of reagents and haemoglobin, allowing for precise and reproducible HbA1c determination by preventing denaturation and precipitation, and stabilising leuco dyes to enhance measurement accuracy.
Implementation Method 1
bringing the haemoglobin, including HbA1c, released in method step a) into contact with a proteolytically acting agent for producing glycated haemoglobin degradation products
Implementation Method 2
The fructosyl amino acid or fructosyl peptide is oxidised by the activity of the enzyme fructosyl amino acid oxidase (FAOX), wherein a result of that oxidation step is the production of hydrogen peroxide (H2O2)
Implementation Method 3
a colorless leuco dye is oxidised with hydrogen peroxide in the presence of a peroxidase to a colored form
Data Source
AI summary
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.