Human in-cell scFv library from infiltrating B cell.

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• Traditional methods for creating single-chain variable fragment (scFv) antibody libraries involve random heavy and light chain assortment.
• This random assortment is not ideal for studying antibody repertoires formed in vivo, particularly during autoimmune diseases.

Purpose of the Study:

• To develop a method for in situ pairing of immunoglobulin heavy (VH) and light (VL) variable region genes in human B cells.
• To enable the study of antibody repertoires and VH/VL pairing in the context of disease processes.

Main Methods:

• Utilized in-cell polymerase chain reaction (PCR) and Cre-recombination in human B cells (CD19+).
• Isolated VH and VL genes from human tissue, amplified them using nested primers, and performed three rounds of PCR with Cre-loxP recombination.
• Cloned the resulting 800-bp band corresponding to scFv and selected human scFv fragments.

Main Results:

• Successfully achieved in situ amplification and association of VH and VL genes within human B cells.
• Generated and selected human scFv fragments using the developed in-cell procedure.

Conclusions:

• The described in-cell amplification, association, and scFv selection procedure is a valuable tool.
• This method offers potential for studying antibody repertoire and VH/VL pairing during disease development.