IN-CELL ASSEMBLY OF SCFV FROM HUMAN THYROID-INFILTRATING B-CELLS

The construction of a large library of single-chain Fv (scFv) antibody fragments involves a random assortment of heavy and light chains. Alt hough useful for the production of recombinant antibodies, this method is not adapted to the study of the autoantibody repertoire formed in vivo during autoimmune diseases. To attain this objective, we describe the use of the in-cell PCR together with Cre-recombination applied, t o our knowledge, for the first time 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 rec ombination of the VH and VL genes within CD19(+) B cells isolated from human thyroid tissue. Nested primers were designed to amplify the kno wn major human VH and VL gene families. After reverse transcription PC R and three rounds of PCR including recombination between VH and VL us ing the Cre-loxP system, we obtained a unique 800-bp band correspondin g in size to scFv fragments. We provide evidence that recombination be tween VH and VL genes occurred inside the same cell. This in-cell ampl ification and association procedure is a potentially useful tool for t he study of autoantibody gene families and the VH/VL pairing that occu rs during the autoimmune process.