Creation of recombinant antigen-binding molecules derived from hybridomas secreting specific

t his protocol describes the design and development of recombinant monovalent antigen-binding molecules derived from monoclonal antibodies through rapid identification and cloning of the functional variable heavy ( VH) and variable light ( VL) genes and the design and cloning of a synthetic D na sequence optimized for expression in recombinant bacteria. t ypically, monoclonal antibodies are obtained from mouse hybridomas, which most often result from the fusion of B lymphocytes from immunized mice with murine myeloma cells. t he protocol described here has previously been exploited for the successful development of multiple antibody-based molecules targeting a wide range of biomolecular targets. t he protocol is accessible for research groups who may not be specialized in this area, and should permit the straightforward reverse engineering of functional, recombinant antigen-binding molecules from hybridoma cells secreting functional IgGs within 50 working days. Furthermore, convenient strategies for purification of antibody fragments are described.