Multimeric Protein Complexes for Antigen-Specific B Cell Isolation
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Solution Overview
Problem
Current methods for characterizing and isolating B cells responsive to specific antigens are limited by the need for complex isolation and identification processes, particularly in identifying cells that express desired receptors for particular antigens.
Innovation Solution
The use of a self-assembling multimeric protein structure with a capture tag and complementary affinity sequence allows for the formation of a complex that binds to target proteins, enabling efficient isolation of immune cells such as B cells through magnetic or fluorescent means, using nucleic acids and kits that include precursors to these complexes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional methods are used to isolate and identify B cells expressing specific antigen receptors, then the process is highly complex and time-consuming, but the isolation efficiency and precision are limited
Solution Approach 1:
The complex isolation process is segmented into distinct functional modules: (1) multimeric protein complex formation with antigen display, (2) fluorescent labeling for detection, and (3) magnetic bead-based separation. Each module performs a specific function, allowing parallel optimization and simplifying the overall process while maintaining high efficiency and precision for isolating antigen-specific B cells
Solution Approach 2:
The patent introduces multimeric protein complexes as intermediary structures that bridge the antigen and B cell receptors. These complexes serve as mediators by simultaneously binding multiple antigen molecules and presenting them to B cells, thereby enhancing the isolation efficiency without requiring complex procedural steps
2Measurement precision
If existing tetramer systems are used to identify antigen-specific cells, then the identification capability is limited, but the fluorescence intensity and detection sensitivity are insufficient
Solution Approach 1:
The patent employs composite multimeric protein structures that combine multiple functional elements: antigen-binding domains, fluorescent protein tags, and multimerization domains. This composite structure achieves both high detection sensitivity and intense fluorescence signal by integrating multiple copies of fluorescent proteins around the antigen, thereby simultaneously improving measurement precision and illumination intensity
Solution Approach 2:
The patent transitions from traditional monomeric or dimeric antigen presentation to multimeric complexes with multiple antigen copies arranged in three-dimensional structures. This dimensional expansion increases the signal-to-noise ratio by presenting multiple antigen epitopes simultaneously, thereby enhancing both detection sensitivity and fluorescence intensity through spatial multiplication
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 efficient recognition and isolation of immune cells, including B cells, with enhanced fluorescence intensity for identification and purification, surpassing existing tetramer systems in identifying antigen-specific cells.
Implementation Method 1
The multimeric protein structure is an assembled complex of monomeric protein substructures. The monomeric protein substrucutres can self-assemble to form the multimeric protein structure.
Implementation Method 2
The monomeric protein substructure is further fused with a complementary affinity sequence. The complementary affinity sequence can then bind to beads affixed with the complementary binding partner to the complementary affinity sequence, thereby attaching the complex to a solid support.
Implementation Method 3
In cases where beads are utilized, the presence of ferromagnetic material in the bead provides a further option to isolate the beads by application of a magnetic field.
Implementation Method 4
The monomeric protein substructure may further be fused with a fluorophore to render the complex visible and also provide a further mechanism for isolating cells associated with the complex, such as by flow cytometry.
Data Source
AI summary
Provided are compositions and methods for use in isolating cells responsive to a target protein by first contacting a collection of isolated cells in an in vitro sample to a complex and then isolating the complex. The complex is formed from a target protein with a capture tag coupled to a multimeric protein structure of at least two self-assembled copies of a monomeric protein substructure fused with a capture sequence.