Antibody Variable Gene Cloning via Ligation-Independent Insertion
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Solution Overview
Problem
Current methods for isolating and producing antibodies, such as hybridoma technology and previous approaches, are inefficient and require intermediate cloning and analysis steps, which are time-consuming and labor-intensive, especially when dealing with B-cells from immunized animals like rabbits.
Innovation Solution
A method that synthesizes single-stranded cDNA from antibody-secreting B-cells using RT-PCR, amplifies variable domain encoding nucleic acids, and inserts them directly into eukaryotic expression plasmids without intermediate cloning or analysis, using ligation-independent cloning for efficient antibody production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If hybridoma technology is used to obtain cells secreting monoclonal antibodies, then monoclonal antibodies can be produced, but only a fraction of B-cells can be fused and propagated, resulting in low efficiency and time-consuming processes
Solution Approach 1:
The invention extracts and utilizes the nucleic acid material directly from single B-cells without requiring cell fusion or propagation. By isolating RNA from individual B-cells and converting it to cDNA, the method bypasses the time-consuming hybridoma formation process while maintaining the ability to produce monoclonal antibodies.
Solution Approach 2:
The invention introduces nucleic acid amplification (RT-PCR) as an intermediary step between B-cell isolation and antibody production. This intermediary process allows the genetic information from a single B-cell to be amplified and expressed in heterologous cells, dramatically improving productivity without requiring the original B-cell to be propagated.
2Manufacturing precision
If intermediate cloning and analysis steps are performed for antibody isolation, then proper antibody sequences can be verified, but the process becomes labor-intensive and time-consuming
Solution Approach 1:
The invention employs self-service through the use of molecular biology techniques that automatically verify and propagate correct sequences. The RT-PCR amplification and subsequent cloning into expression vectors with selectable markers allow the system to self-verify correct inserts and self-propagate successful clones without extensive manual analysis.
Solution Approach 2:
The invention replaces manual mechanical operations (manual cloning, plasmid isolation, sequencing verification) with automated molecular biology processes. PCR amplification, automated cloning techniques, and high-throughput sequencing replace labor-intensive manual steps, improving ease of operation while maintaining precision.
3Adaptability or versatility
If B-cells from rabbits are used for antibody production, then diverse antibodies can be obtained, but these B-cells produce low antibody yields making traditional methods ineffective
Solution Approach 1:
The invention performs preliminary action by isolating and amplifying the nucleic acid material from single B-cells before antibody expression. RT-PCR amplification of the variable region genes from rabbit B-cells creates sufficient genetic material for expression in heterologous systems, overcoming the low natural antibody yield of rabbit B-cells.
Solution Approach 2:
The invention uses copying by expressing the amplified nucleic acid sequences from rabbit B-cells in heterologous host cells (such as CHO or HEK293 cells). These host cells serve as copies that can produce high yields of the antibody while maintaining the original antibody sequence and specificity from the rabbit B-cell.
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 significantly reduces the time and labor required for antibody production, allowing for faster isolation and expression of diverse antibodies with high affinity, and is particularly effective for B-cells that produce low antibody yields, such as those from rabbits.
Implementation Method 1
synthesizing single stranded cDNA using the RNA obtained from an antibody secreting B-cell as template in an RT-PCR
Implementation Method 2
amplifying the variable domain encoding nucleic acids in a PCR
Data Source
AI summary
In the method as reported herein the isolation of nucleic acid segments encoding antibody variable domains and the insertion of the isolated nucleic acid segments in eukaryotic expression plasmids is performed without the intermediate isolation and analysis of clonal intermediate plasmids. Thus, in the method as reported herein the intermediate cloning, isolation and analysis of intermediate plasmids is not required, e.g. by analysis of isolated transformed E. coli cells.