Dried Blood Spot Enzyme Assay for Lysosomal Storage Disease Screening
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Solution Overview
Problem
Current methods for detecting enzyme deficiencies associated with lysosomal storage diseases, such as Gaucher, Fabry, Krabbe, Niemann-Pick A/B, and Pompe diseases, are not robust or reliable enough for early presymptomatic detection, particularly in newborns, which limits the effectiveness of early therapeutic interventions.
Innovation Solution
A method involving the extraction of enzymes from dried blood spots using specific buffer solutions, followed by the addition of substrate, internal standards, and inhibitors, with precise molar ratios, and subsequent purification and quantification using tandem mass spectrometry to determine enzyme activity, allowing for the identification of decreased enzyme levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If mass spectrometry is used to determine enzyme activity from dried blood spots, then detection capability is provided, but the methods lack robustness and reliability for early presymptomatic detection
Solution Approach 1:
The patent applies parameter changes by optimizing multiple assay conditions including buffer composition (pH, ionic strength), substrate concentrations, incubation temperature, and incubation time. These parameter optimizations transform the detection method to achieve both high reliability for presymptomatic detection and high measurement precision for enzyme activity quantification
Solution Approach 2:
The patent implements preliminary action through extensive method development and validation performed before clinical application. This includes optimizing extraction protocols, selecting appropriate internal standards, establishing quality control procedures, and validating the assay across multiple laboratories to ensure robustness and reliability before newborn screening implementation
2Loss of time
If early presymptomatic detection is implemented, then treatment benefit is maximized, but detection methods must be sufficiently robust and reliable
Solution Approach 1:
The patent performs preliminary optimization of the entire detection workflow including blood spot collection, extraction, assay setup, and data analysis before clinical implementation. This preliminary action ensures the method is robust and reliable for early presymptomatic detection, allowing rapid screening without compromising accuracy
Solution Approach 2:
The patent optimizes parameters such as extraction buffer composition, incubation conditions, and mass spectrometry settings to enable rapid yet reliable detection. These parameter changes allow the assay to be completed quickly while maintaining high reliability for presymptomatic diagnosis
3Adaptability or versatility
If multiple enzyme activities are determined simultaneously, then comprehensive screening is achieved, but assay complexity increases
Solution Approach 1:
The patent merges multiple enzyme activity assays into a single unified platform using tandem mass spectrometry. By combining the detection of acid glucosidase, acid sphingomyelinase, galactocerebroside beta-galactosidase, and other lysosomal enzymes in one assay, the patent achieves comprehensive screening while managing complexity through standardized protocols and automated analysis
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 method provides a robust and reliable means for early detection of enzyme deficiencies, enabling timely therapeutic interventions and improving treatment outcomes for newborns by accurately quantifying acid glucocerebrosidase, acid sphingomyelinase, galactocerebroside β-galactosidase, and other enzyme activities.
Implementation Method 1
extracting acid glucosidase from a dried blood spot obtained from the subject using an aqueous buffer solution
Implementation Method 2
extracting acid glucosidase from a dried blood spot obtained from the subject using an aqueous buffer solution
Implementation Method 3
quenching the reaction by addition of four volumes of a solution consisting essentially of ethyl acetate/methanol (1/1)
Implementation Method 4
extracting the reaction product and internal standard by adding at least about a 3/5 volume of ethyl acetate and at least about a 3/5 volume of water to the quenched reaction to form a two-phase system
Implementation Method 5
purifying the reaction product and internal standard by passage of the extract from (e) through silica gel
Implementation Method 6
purifying the reaction product and internal standard by passage of the extract from (e) through silica gel
Data Source
Figure 1A
Figure 1B
Figure 1C
AI summary
The present invention provides compositions for performing assays of enzyme activity associated with lysosomal storage diseases. The invention further provides methods for determining enzyme activity, and methods for the screening for lysosomal storage disease in an individual.