LRP5 Methylation Analysis for B Cell Identification
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Solution Overview
Problem
Current methods for identifying and quantifying B cells in complex samples, such as blood or immune cells, are not robust enough and often require purification or enrichment steps, limiting their reliability and convenience.
Innovation Solution
Analyzing the methylation status of CpG positions in the LRP5 gene region, specifically using bisulfite convertibility to differentiate B cells from other cells, allowing for their identification and quantification without purification, using specific primers and amplification methods like PCR.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current methods are used to identify and quantify B cells in complex samples, then B cells can be detected, but the methods lack robustness and require purification or enrichment steps
Solution Approach 1:
The patent changes the detection parameter from protein-based markers (requiring purification) to epigenetic methylation status of LRP5 gene CpG positions. This parameter change enables direct detection in complex samples without purification steps, as methylation patterns are cell-type specific and can be analyzed directly from total DNA extracted from whole blood or tissue samples.
Solution Approach 2:
The patent replaces mechanical separation methods (purification and enrichment steps) with a molecular detection method based on bisulfite conversion and PCR amplification. Instead of physically isolating B cells through flow cytometry or magnetic separation, the method uses chemical conversion of methylated cytosines followed by specific amplification of demethylated LRP5 gene regions, eliminating the need for complex purification equipment and procedures.
2Measurement precision
If purification steps are performed to improve B cell identification accuracy, then specificity increases, but the process becomes more complex and time-consuming
Solution Approach 1:
The patent uses epigenetic methylation status as a specific parameter that is inherently different between B cells and other cell types. The LRP5 gene shows characteristic demethylation in B cells compared to methylation in other cells, providing natural specificity without requiring time-consuming purification steps to separate B cells from other immune cells.
Solution Approach 2:
The patent performs preliminary bisulfite conversion of the DNA sample before amplification, which chemically modifies methylated cytosines to uracils while leaving unmethylated cytosines unchanged. This preliminary chemical action creates sequence differences that allow subsequent PCR primers to specifically amplify only demethylated LRP5 regions from B cells, achieving specificity without physical separation.
3Adaptability or versatility
If conventional identification methods are used, then B cells can be detected, but the methods are not suitable for direct analysis in whole blood and non-trypsinized tissue
Solution Approach 1:
The patent develops a universal detection method based on epigenetic analysis that works across multiple sample types including whole blood, non-trypsinized tissue, and other complex biological samples. The methylation-specific PCR approach is universally applicable because it targets fundamental epigenetic differences between cell types that exist in all these sample types, eliminating the need for sample-specific optimization.
Solution Approach 2:
The patent uses total DNA extraction as an intermediary step that converts diverse sample types (whole blood, tissue, etc.) into a common format suitable for bisulfite conversion and PCR analysis. This intermediary DNA extraction step harmonizes different sample types, allowing the same methylation-specific protocol to be applied reliably across all sample types without requiring sample-specific processing.
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 to detect and quantify B cells, distinguishing them from other immune cells with high specificity, enabling applications in diagnostics and monitoring of autoimmune diseases, cancers, and immune status assessment.
Implementation Method 1
analyzing the methylation status of at least one cytosine-phosphodiester-guanine (CpG) position in the mammalian gene region for Low density lipoprotein receptor-related protein 5 (LRP5), wherein a demethylation or lack of methylation of said gene region is indicative for a B cell
Data Source
AI summary
The present invention relates to a method, in particular an in vitro method, for identifying B cells, comprising analyzing the methylation status of at least one CpG position in the mammalian gene region for Low density lipoprotein receptor-related protein 5 (LRP5), wherein a demethylation or lack of methylation of said gene region is indicative for a B cell, when compared to a non-B cell. The analyses according to the invention can identify B cells on an epi-genetic level and distinguish them from all other cells in complex samples, such as, for example, other blood or immune cells. The present invention furthermore provides an improved method for quantifying B cells, in particular in complex samples. The method can be performed without a step of purifying and/or enriching cells, preferably in whole blood and/or non-trypsinized tissue. Further claimed are kits and specific primers and probes for identifying methylation.