title: Creation of recombinant antigen-binding molecules derived from hybridomas secreting specific antibodies.
abstract: This 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 DNA sequence optimized for expression in recombinant bacteria. Typically, monoclonal antibodies are obtained from mouse hybridomas, which most often result from the fusion of B lymphocytes from immunized mice with murine myeloma cells. The protocol described here has previously been exploited for the successful development of multiple antibody-based molecules targeting a wide range of biomolecular targets. The 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.
authors.affiliation: UCD Centre for Nanomedicine, School of Chemistry and Chemical Biology, University College Dublin, Dublin, Ireland.
authors.forename: Conor
authors.initials: C
authors.lastname: Fields
authors.forename: David
authors.initials: D
authors.lastname: O'Connell
authors.forename: Sujing
authors.initials: S
authors.lastname: Xiao
authors.forename: Gil U
authors.initials: GU
authors.lastname: Lee
authors.forename: Philippe
authors.initials: P
authors.lastname: Billiald
authors.forename: Julien
authors.initials: J
authors.lastname: Muzard
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D003001:Cloning, Molecular; D006825:Hybridomas / Q000737:chemistry / Q000378:metabolism*; D007128:Immunoglobulin Fragments / Q000302:isolation & purification*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D051379:Mice; D008958:Models, Molecular; D011995:Recombination, Genetic
chemical_list: D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains