Human in-cell scFv library from infiltrating B cell.
The construction of a large library of single-chain Fv (scFv) antibody fragments involves a random assortment of heavy and light chains. Although useful for the production of recombinant antibodies, this method is not totally adapted to the study of the antibody repertoire formed in vivo during, for example, autoimmune diseases.We describe here, the use of the in-cell PCR together with Cre-recombination applied to human B cells to obtain in situ pairing of the variable (V) region genes of the immunoglobulin heavy (H) and light (L) chains. Our method is based on amplification and recombination of the VH and VL genes within CD19+ B cells isolated from human tissue. Nested primers were designed to amplify the known major human VH and VL gene families. After reverse transcription PCR and three rounds of PCR including recombination between VH and VL using the Cre-loxP system, the 800-bp band corresponding to scFv was cloned and human scFv fragments selected.This in-cell amplification, association, and scFv selection procedure is a potentially useful tool for the study of antibody repertoire and the VH/VL pairing that occurs during the diseases' process.
CPID, UMR 5232, CNRS, Montpellier, France. sylvie.roux@univ-montp1.fr
Sylvie
S
Peraldi-Roux
D001402:B-Lymphocytes / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002469:Cell Separation; D006801:Humans; D019426:Integrases / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000235:genetics; D048348:Reverse Transcription / Q000235:genetics; D057127:Single-Chain Antibodies / Q000378:metabolism*
D019151:Peptide Library; D057127:Single-Chain Antibodies; C045073:Cre recombinase; D019426:Integrases