Production of monoclonal recombinant antibodies without the use of hybridomas by (in vitro) spleen fragment culture combined with isothermal self-sustained sequence replication of rna

摘要

The invention is directed to a method for producing a recombinant protein having a preselected binding affinity and specificity for a given antigen. Generally, the method combines spleen fragment culture for the in vitro somatic mutation of B cells in response to antigen, followed by preferential amplification of RNA encoding heavy and light chains of desired antibodies produced by those B cells. The amplified RNA is then converted to DNA and incorporated into expression vectors; transfected host cells then express the antibody chains or a single chain antibody having preselected characteristics. Also disclosed are generic degenerate primer pools for the efficient amplification of RNA encoding mouse IgG.