Cloning antibodies from single cells in pooled sequence libraries by selective PCR.

Area of Science:

• Immunology
• Molecular Biology
• Biotechnology

Background:

• Antibody discovery relies on determining antigen binding characteristics, necessitating antibody cloning and expression.
• Current high-throughput antibody sequencing methods pool DNA from multiple cells, hindering single-cell antibody cloning.
• Cloning antibodies from single B cells is crucial for understanding their specific binding properties.

Purpose of the Study:

• To present a novel strategy for retrieving and cloning antibody DNA from individual cells within a pooled library.
• To enable high-throughput functional characterization of native antibodies by facilitating single-cell cloning.
• To overcome limitations of current methods that obscure single-cell antibody origins.

Main Methods:

• Development of Selective PCR for Antibody Retrieval (SPAR), a method utilizing unique sequence barcodes on cDNA molecules.
• Leveraging sequence barcodes for specific amplification of antibody heavy- and light-chain cDNA from single cells via PCR.
• Computational analysis to predict the retrieval efficiency of SPAR for typical high-throughput sequencing data.

Main Results:

• SPAR successfully retrieves full-length antibody variable region cDNA from single cells within large pooled libraries (~5,000 cells).
• Computational analysis indicates SPAR can retrieve most human antibodies sequenced by standard high-throughput methods.
• Experimental validation demonstrates the feasibility of SPAR for isolating antibody DNA from individual B cells.

Conclusions:

• SPAR offers a rapid, low-cost solution for cloning and expressing native human antibodies from pooled single-cell sequence libraries.
• This technique facilitates the functional characterization of antibodies derived from single B cells.
• SPAR advances antibody discovery and engineering by enabling precise single-cell-based cloning.