A NEW METHOD FOR THE ANALYSIS AND PRODUCTION OF MONOCLONAL-ANTIBODY FRAGMENTS ORIGINATING FROM SINGLE HUMAN B-CELLS

The phage display approach has proven to be a major step forward in st udies on the human autoimmune repertoire. However, it remains doubtful whether the heavy and light chains of the antibodies obtained from th ese libraries resemble original in vivo pairings. Here we describe a n ovel, simple method for the immortalization of the variable heavy and light chain regions originating from individual, nonboosted, autoantig en-specific human B cells. Our method is based on the clonal expansion of B cells in which cell-cell interactions (CD40-CD40L) as well as so luble factors were shown to be essential. This B cell culture system c ombined with a selection on antigen (the U1A protein, a frequent autoa ntigenic target in patients with systemic lupus erythematosus) and sin gle cell sorting resulted in the isolation of U1A-specific human B cel ls and the subsequent expression of an U1A-specific single chain varia ble fragment (scFv). Our method circumvents laborious plating and scre ening and has the advantage that original heavy/light chain pairings c an be isolated. Due to the high growth efficiency of single cultured B cells (50-70% outgrowth) even rare B cell activities can be studied u sing this system. (C) 1997 Elsevier Science B.V.