Cloning and expression of murine Ig genes from single B cells

Area of Science:

• Immunology
• Molecular Biology
• Biotechnology

Background:

• Characterizing the antibody repertoire is crucial for understanding immune responses.
• Existing methods for antibody repertoire analysis can be inefficient or biased.
• High-throughput methods are needed to analyze the diversity of antibody genes.

Purpose of the Study:

• To develop a highly efficient 96-well format strategy for characterizing the murine antibody repertoire.
• To combine immunoglobulin (Ig) gene cloning with single-cell antibody expression and reactivity profiling.
• To enable unbiased sequence analysis and parallel reactivity testing of recombinant monoclonal antibodies.

Main Methods:

• Isolation of individual mouse B lineage cells using fluorescence-activated cell sorting (FACS) based on surface marker expression.
• Amplification of full-length Ig heavy (H) and Ig light (L) chain variable (V) region gene transcripts via RT-PCR.
• Cloning amplified products into eukaryotic expression vectors for in vitro production of monoclonal antibodies.

Main Results:

• Successful in vitro production of monoclonal antibodies with antigen specificities matching the original B cell antigen receptors.
• Obtained IgH and IgL chain gene sequence information directly linked to recombinant antibody reactivity profiles.
• Demonstrated a highly efficient and unbiased method for antibody repertoire characterization.

Conclusions:

• The established RT-PCR based strategy enables efficient and unbiased characterization of the expressed murine antibody repertoire.
• This method allows for parallel sequence analysis and antibody reactivity testing at the single-cell level.
• The approach facilitates a comprehensive understanding of antibody diversity and function.