Enzymatic Blood Assay Endpoint Estimation
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Solution Overview
Problem
Current point-of-care assays for measuring lipid components in blood samples, particularly those associated with specific lipoproteins, face challenges such as the need for calibrators, instability of enzymes, and temporary blocking methods that prevent accurate end-point measurements, leading to longer assay times and higher costs due to the use of dried reagents.
Innovation Solution
A method using liquid reagents that allows for the estimation of an unmeasurable end-point in enzymatic reactions, enabling the simultaneous measurement of multiple analytes with reduced assay time and without the need for calibrators, by monitoring the reaction until at least 50% of the target analyte is converted and applying suitable algorithms to predict the end-point.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If end-point assays are used for lipid components with temporary blocking, then measurement accuracy is improved, but assay time increases and reliability deteriorates due to unmeasurable endpoints
Solution Approach 1:
The blocking reagent is added to the sample before the enzymatic reaction to prevent interference from non-HDL cholesterol. This preliminary blocking action ensures that when the enzymatic reaction occurs, only HDL cholesterol is measured, providing accurate endpoint measurements without requiring extended assay times to resolve interference.
Solution Approach 2:
A blocking reagent is introduced as an intermediary substance that temporarily prevents non-HDL cholesterol from interfering with the enzymatic measurement. This intermediary blocks the unwanted reaction pathways while allowing the desired HDL cholesterol measurement to proceed accurately and efficiently.
2Reliability
If dried reagents are used to ensure stability, then reliability is improved, but cost increases and ease of operation deteriorates due to reconstitution requirements
Solution Approach 1:
The patent changes the physical state of the reagents from dried to liquid form. Liquid reagents maintain stability through proper formulation and storage conditions without requiring reconstitution, thereby reducing operational complexity and cost while maintaining reliability. The enzymatic reaction components are formulated as stable liquid solutions that can be used directly in the assay.
3Measurement precision
If calibrators are used to compensate for enzyme inactivation, then measurement accuracy is improved, but device complexity and cost increase
Solution Approach 1:
The assay system performs self-validation through internal controls and quality checks built into the liquid reagent formulation. The reagents are designed to maintain consistent activity throughout the assay without requiring external calibrators or complex compensation mechanisms. The system monitors reaction progress and automatically adjusts for enzyme activity variations through the liquid reagent's stable formulation.
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
This approach enables faster, cost-effective, and accurate measurement of lipid components in blood samples, reducing assay time and eliminating the reliance on calibrators and dried reagents, while maintaining the reliability of end-point analysis.
Implementation Method 1
contacting said blood cell containing sample with a reagent mixture that dilutes the sample
Implementation Method 2
substantially removing blood cells to provide a substantially cell free sample
Implementation Method 3
contacting said sample with at least one reagent which serves to temporarily and/or competitively prevent reaction of said second component, whereby to generate a blocked second component
Implementation Method 4
contacting said sample with at least one reagent mixture comprising at least one converting enzyme for at least one constituent of each analyte of said at least one analyte, whereby to cause the selective reaction of the or each analyte to directly or indirectly generate detectable reaction products
Implementation Method 5
relating an amount of said detectable product or products and/or a rate of formation of said detectable product or products to the concentration of the or each of said at least one analyte in said blood sample, wherein the concentration of at least said first component is related to a corresponding detectable reaction product by means of estimating an unmeasurable (fictive) endpoint
Data Source
AI summary
The invention provides an enzymatic method for measuring the concentration of one or more analytes in the plasma portion of a blood derived sample, containing a first and a second component, where said second component interferes with the measurement of said first component. The method includes: i) diluting the sample with a reagent mixture; ii) substantially removing blood cells; iii) using a reagent which serves to temporarily prevent reaction of the second component, to generate a blocked second component; iv) causing the selective reaction of a constituent of each analyte to directly or indirectly generate detectable reaction products, where one of the analytes is the first component; v) monitoring the detectable reaction product or products; vi) relating an amount of the detectable product or products and/or a rate of formation of the detectable product or products to the concentration of each analyte, where the concentration of at least the first component is related to a corresponding detectable reaction product by means of estimating an un-measurable (fictive) endpoint. Step iii) may be carried out at any stage up to and including step iv) but before steps v) or vi). The reagent of step iii) may be applied to the sample separately or may be included in a reagent mixture during steps i) or iv). A corresponding kit is also provided.