Multiplexed PCR for Single-Cell Immunoglobulin Amplification
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Solution Overview
Problem
Current methods for generating monoclonal immunoglobulins, such as hybridoma technology and phage display, face challenges such as limited diversity, cross-reactivity, and inefficient selection of high-affinity immunoglobulin pairs.
Innovation Solution
A method involving multiplexed polymerase chain reaction (PCR) and in vitro translation to obtain and produce human monoclonal antibodies from a single immunoglobulin-producing cell, allowing for the characterization of antigen binding characteristics and avoiding loss of immunoglobulin diversity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If hybridoma technology is used to generate monoclonal immunoglobulins, then stable clones can be obtained, but diversity of the antibodies is limited because only a limited number of B-cells are successfully fused, propagated and characterized
Solution Approach 1:
The invention extracts and amplifies nucleic acid sequences encoding immunoglobulin variable regions from individual B-cells or plasma cells, isolating the genetic information without requiring cell fusion. This allows recovery of diverse antibody sequences from multiple single cells while maintaining stability through molecular cloning and expression in host cells
Solution Approach 2:
The invention creates molecular copies of immunoglobulin encoding nucleic acids through PCR amplification and cloning into expression vectors. These copied sequences can be expressed in host cells to produce monoclonal antibodies, preserving the diversity of original B-cell repertoire while enabling stable propagation
2Productivity
If phage or yeast display-based combinatorial library approaches are used, then large numbers of immunoglobulin pairs can be generated, but cross-reactivity occurs due to random pairing of heavy and light chains
Solution Approach 1:
The invention extracts nucleic acid sequences encoding immunoglobulin heavy and light chains from individual B-cells or plasma cells that have already formed correct cognate pairs in the organism. This extraction approach preserves the natural pairing specificity while allowing high-throughput recovery of functional antibody sequences without random pairing artifacts
Solution Approach 2:
The invention uses nucleic acid sequences as intermediaries to reconstruct immunoglobulin pairs. By amplifying and reassembling the genetic information from single cells, the method maintains the specific heavy-light chain pairings that occurred during normal immune development, avoiding the cross-reactivity problems of random combinatorial libraries
3Reliability
If combinatorial libraries are screened to identify high-affinity immunoglobulin pairs, then selection pressure can be applied, but genetic diversity is limited due to inherent selection biases
Solution Approach 1:
The invention extracts nucleic acid sequences from individual B-cells or plasma cells that have already undergone in vivo selection for high affinity, preserving the diversity of naturally selected antibodies. By directly recovering sequences from functional single cells rather than screening random combinations, the method maintains genuine genetic diversity while ensuring high affinity through the natural selection that already occurred in the organism
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 enables the efficient generation of monoclonal antibodies with high specificity and diversity, reducing cross-reactivity and allowing for the production of functional Fab fragments.
Implementation Method 1
A method involving multiplexed polymerase chain reaction (PCR) and in vitro translation to obtain and produce human monoclonal antibodies from a single immunoglobulin-producing cell
Implementation Method 2
A method involving multiplexed polymerase chain reaction (PCR) and in vitro translation to obtain and produce human monoclonal antibodies from a single immunoglobulin-producing cell
Data Source
AI summary
The current invention is directed to a method for obtaining a nucleic acid encoding an immunoglobulin variable domain from a single cell comprising the following steps:Performing a first polymerase chain reaction with three to six 5′-primers and one 3′-primer,Performing with the product of the first polymerase chain reaction a second polymerase chain reaction with thirteen to sixteen primers and one 3′-primer,whereby the distance of the binding locations of the primer employed in the second polymerase chain reaction is reduced compared to the first polymerase chain reaction.