Immunoglobulin Isotype Profiling via Massively Parallel Sequencing
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Solution Overview
Problem
Current methods for diagnosing and monitoring autoimmune diseases and other immune-related conditions face challenges in distinguishing between diverse immune cell sequences, often requiring specific cell populations and spatial isolation, which limits their ability to detect subtle clonal expansions and disease-specific signatures.
Innovation Solution
A non-invasive method involving the isolation and amplification of RNA nucleic acids from a heterogeneous biological sample, followed by massively parallel sequencing to generate immunoglobulin isotype profiles, allowing for the detection and characterization of specific isotype sequences indicative of disease states such as autoimmune diseases, infectious diseases, or cancer.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If spectratyping is used to detect clonal expansion, then specific V segment sequences can be amplified and visualized, but many distinct sequences with the same length become indistinguishable, limiting detection sensitivity
Solution Approach 1:
The patent changes the detection parameter from length-based (spectratyping) to sequence-based (massively parallel sequencing). By using massively parallel sequencing, the invention can distinguish sequences based on their actual nucleotide composition rather than just length, thereby resolving the contradiction between detection sensitivity and sequence discrimination.
Solution Approach 2:
The patent replaces the mechanical gel electrophoresis separation system with a computational sequencing and analysis system. This substitution allows for direct sequence reading and comparison, eliminating the information loss inherent in length-based separation methods while maintaining high detection sensitivity.
2Reliability
If standard immunological techniques are used to detect autoantibodies, then circulating autoantibodies can be identified, but the methods cannot distinguish whether the antibodies themselves are pathological or if the targets are the same as those tested in vitro
Solution Approach 1:
The patent extracts and sequences the specific nucleic acid sequences encoding the variable regions of immunoglobulins. By directly sequencing the B-cell receptor genes, the invention obtains information about the actual immune repertoire and clonal expansions, providing more reliable diagnostic information than indirect autoantibody detection.
Solution Approach 2:
The patent uses massively parallel sequencing as an intermediary to bridge the gap between detecting autoantibodies and understanding their pathological significance. The sequencing data provides direct information about B-cell clonal expansions and immune repertoire changes, serving as a mediator that connects antibody detection with pathological context.
3Measurement precision
If spectratyping is used to assess clonal expansion, then V segment amplification can be performed, but the technique requires dramatic clonal expansion to be discernible, missing subtle disease signatures
Solution Approach 1:
The patent changes the detection threshold parameter by using massively parallel sequencing, which can detect and quantify even minor clonal expansions through direct sequence counting. This allows for the detection of subtle clonal expansions that would be invisible in spectratyping, while maintaining the ability to detect dramatic expansions.
4Measurement precision
If methods requiring specific cell populations and spatial isolation are used, then individual cell analysis can be performed, but the complexity of the procedure increases and smaller sample sizes cannot be effectively utilized
Solution Approach 1:
The patent merges the analysis of multiple cells by using massively parallel sequencing on bulk RNA or DNA samples. The high-throughput nature of the sequencing allows individual cell transcripts to be distinguished through unique molecular identifiers or by analyzing the diversity of sequences, thereby achieving individual cell resolution without the need for physical cell isolation or complex single-cell processing.
Solution Approach 2:
The patent creates a universal method that works with heterogeneous cell populations without requiring specific cell isolation. The massively parallel sequencing approach can analyze total RNA or DNA from mixed samples, making the method universally applicable to various sample types and eliminating the need for complex sample preparation procedures.
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 the generation of detailed immunoglobulin isotype profiles from a small sample size, such as a single drop of blood, without the need for trained phlebotomy or fractionation, providing high sensitivity and specificity in diagnosing and monitoring immune system disorders, including autoimmune diseases and cancer.
Implementation Method 1
isolating and amplifying a plurality of RNA nucleic acids from the biological sample
Implementation Method 2
amplifying a plurality of RNA nucleic acids from the biological sample, wherein the RNA comprises Ig isotype constant region sequences, to generate amplicons
Implementation Method 3
sequencing the amplicons in a massively parallel sequencing reaction using a sequencing technique that generates at least 10,000 sequence reads per run
Implementation Method 4
analyzing sequence data from (b) by extracting Ig constant region sequences characteristic of each isotype, and comparing the number of isotype sequences for each isotype
Data Source
Figure 1
AI summary
The invention provides a non-invasive technique for the detection and quantification of immune globulin isotypes, in a biological sample containing a plurality of distinct cell populations. Methods are conducted using sequencing technology to detect and enumerate immunoglobulin isotype profiles within a heterogeneous biological sample.