High-Affinity Antibody Cell Selection by FACS Screening
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Solution Overview
Problem
Existing methods for generating high-affinity monoclonal antibodies are inefficient and time-consuming, particularly due to throughput limitations in hybridoma culture and the need for multiple rounds of screening or site-directed mutagenesis.
Innovation Solution
A method to select and enrich high-affinity antibody-producing cells directly from a population using fluorescence-activated cell sorting (FACS) based on their binding properties, eliminating the need for cloning and subsequent affinity maturation steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If hybridoma technology is used to obtain monoclonal antibodies, then antibody production is achieved, but throughput is limited and isolation of rare high affinity antibodies is inefficient
Solution Approach 1:
The patent replaces the mechanical hybridoma fusion and culture system with a display technology system where antibodies are expressed on the surface of phage, yeast, or mammalian cells. This allows for high-throughput screening of antibody libraries without the throughput limitations of hybridoma culture, enabling efficient isolation of rare high affinity antibodies through multiple rounds of panning or titration.
2Manufacturing precision
If display technology is used to produce antibody candidates from libraries, then high-quality protein libraries are obtained, but diversity is limited and multiple rounds of screening are required
Solution Approach 1:
The patent performs preliminary affinity maturation through in vitro mutagenesis on the antibody candidates before final selection. This preliminary action improves the binding affinity of antibodies derived from display libraries, reducing the need for multiple rounds of screening and accelerating the overall process of obtaining high affinity antibodies.
3Reliability
If in vitro mutagenesis-based affinity maturation is performed, then binding affinity is enhanced, but process complexity and time requirements increase
Solution Approach 1:
The patent uses in vitro mutagenesis to introduce specific amino acid changes in the antibody variable regions, thereby changing the binding parameters and enhancing affinity. This controlled parameter change approach allows for systematic improvement of antibody binding characteristics without requiring overly complex procedures, as the mutagenesis is performed on already high-quality candidates from display libraries.
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 allows for the efficient isolation of high-affinity antibodies with binding affinities ranging from 0.1 pM to 10 nM, significantly reducing the number of steps and increasing the probability of obtaining desirable antibodies.
Implementation Method 1
isolated using techniques for single-cell isolation, such as using fluorescence activated cell sorting (FACS)
Implementation Method 2
contacting a population of primary antibody-producing cells with specificity to an antigen of interest with a low concentration of labeled antigen for a time sufficient for the labeled antigen to bind to antibody on the surface of the cells
Data Source
AI summary
The present disclosure relates to a method for identifying cells that express antigen-specific antibodies with a high binding affinity for a monomeric antigen. Using fluorescence activated cell sorting, cells expressing high affinity antigen-specific antibodies are selected from a population of immune cells isolated from a mammal that has been immunized with or otherwise exposed to the antigen. Nucleic acids encoding the high affinity antibodies can then be cloned into other lymphoid and non-lymphoid cells where the antibody can be expressed and from which the antibodies can be secreted.