Antigen-Specific Antibody Identification via ARM-PCR and HTS
Solution Overview
Problem
Current methods for identifying antigen-specific antibodies and T cells are time-consuming, labor-intensive, and lack sensitivity and specificity, often requiring multiple rounds of panning and depending on animal models, which can be immunogenic and ethically challenging, while also struggling to efficiently utilize high-throughput sequencing for immune repertoire analysis.
Innovation Solution
A method combining amplicon rescued multiplex PCR (arm-PCR) with high-throughput immune repertoire sequencing and liquid chromatography tandem mass spectrometry (LC MS/MS) to rapidly identify antigen-specific adaptive immune responses by isolating and comparing amino acid sequences from blood or tissue samples, allowing for cloning and expression of specific antibody sequences that bind to target antigens.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If hybridoma technology is used to produce monoclonal antibodies, then antibody production is achieved, but the process becomes time-consuming and labor-intensive
Solution Approach 1:
The patent replaces manual mechanical processes (multiple rounds of panning, manual isolation, and screening) with automated high-throughput sequencing technology. The arm-PCR method combined with HTS automatically identifies antigen-specific B cells and their antibody sequences, eliminating the need for labor-intensive manual manipulation and significantly reducing the time required for monoclonal antibody identification.
Solution Approach 2:
The invention changes the detection parameters from traditional ELISA-based methods to sequence-based identification using high-throughput sequencing. By sequencing the variable regions of antibody genes directly from single B cells, the method achieves rapid identification of antigen-specific clones without requiring multiple rounds of panning, thus improving productivity while reducing time loss.
2Measurement precision
If multiple rounds of panning are performed in hybridoma or phage display, then antigen-specific clones are identified, but the process becomes increasingly time-consuming and labor-intensive
Solution Approach 1:
The patent performs preliminary enrichment of antigen-specific B cells before sequencing by using antigen-coated magnetic beads or flow cytometry sorting. This preliminary action allows direct identification of specific clones in a single step, eliminating the need for multiple sequential panning rounds while maintaining high identification accuracy through sequence-based verification.
Solution Approach 2:
The invention substitutes repeated mechanical panning operations with a single high-throughput sequencing operation. The arm-PCR method amplifies and sequences antibody gene segments directly from enriched B cell populations, providing accurate identification of antigen-specific clones without requiring multiple manual manipulation steps.
3Productivity
If mouse hybridomas are used for antibody production, then monoclonal antibodies are obtained, but the antibodies have high immunogenicity in humans requiring additional humanization steps
Solution Approach 1:
The patent extracts the variable region genes (VH and VL) directly from human B cells that naturally produce human antibodies. By isolating and sequencing these genes from human donors, the method obtains complete human antibody sequences without requiring any mouse-to-human transfer or humanization engineering, thus eliminating the need for additional complexity in antibody design and production.
Solution Approach 2:
Instead of starting with mouse antibodies and attempting to humanize them, the invention inverts the approach by directly obtaining human antibodies from human B cells. This reverse strategy eliminates the immunogenicity problem entirely, as the antibodies are already human in origin, requiring no further engineering complexity.
4Adaptability or versatility
If phage display libraries are constructed from non-immunized donors, then human antibody libraries are created, but multiple rounds of panning are required for selection
Solution Approach 1:
The patent replaces iterative mechanical panning selection with direct sequence-based identification using high-throughput sequencing. The arm-PCR method sequences the variable regions of B cell receptors from antigen-enriched populations, allowing direct identification of specific clones without requiring multiple rounds of library panning, thus reducing time while maintaining library diversity.
Solution Approach 2:
The invention creates a sequence copy of the antibody variable regions through PCR amplification and sequencing, rather than requiring physical manipulation and selection of phage particles. This molecular copying approach allows rapid identification of specific clones from diverse libraries without the time-consuming iterative panning process.
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
Enables rapid and direct identification of antigen-specific antibodies and T cells, reducing the need for animal models and labor-intensive processes, providing high sensitivity and specificity, and enabling the production of entirely human antibodies with refined affinity for antigens, thus overcoming the limitations of existing technologies.
Implementation Method 1
The invention relates to a method for identifying antibodies that are specific for a given antigenic entity, the method comprising isolating at least one antigen-specific antibody from a blood or tissue sample obtained from a human or animal subject, determining an amino acid sequence (e.g., a peptide sequence) of the at least one antigen-specific antibody
Implementation Method 2
A method combining amplicon rescued multiplex PCR (arm-PCR) with high-throughput immune repertoire sequencing and liquid chromatography tandem mass spectrometry (LC MS/MS)
Implementation Method 3
liquid chromatography tandem mass spectrometry (LC MS/MS) to rapidly identify antigen-specific adaptive immune responses
Data Source
AI summary
Disclosed is a method for correlating at least one amino acid sequence from an antibody isolated from human or animal blood with at least one DNA sequence corresponding to the antibody in the immunorepertoire of the human or animal. The method also provides a means for pairing heavy and light chains to produce synthesized monoclonal antibodies.