engleski

KAC: A novel biological system for the rapid production of site-specific biotinylated monoclonal antibodies

Biotinylation represents one of the most frequently used methods to label monoclonal antibodies. Biotinylation is usually achieved via chemical binding of biotin to random lysine residues, which may interfere with the binding capacity of an antibody when this lysine residue lies within the variable domain. In contrast, enzymatic biotinylation targets only lysine residues within defined peptide sequences. We have exploited enzymatic biotinylation to set up a platform that allows the generation of site-specific biotinylated monoclonal antibodies of any antigen-binding specificity. First, a mouse strain was generated (KAC) that contains a biotin-accepting peptide sequence for the bacterial BirA biotin ligase from E.coli within its kappa light chain. Second, the hybridoma acceptor cell line SP2/O was stably transfected with a modified BirA transgene. This allows for direct biotinylation of antibodies within the ER of these cells after PEG-mediated fusion. KAC mice have an unaltered B cell compartment and responded with normal germinal center responses after infection with mCMV virus or model-antigen immunization. Immunization yielded normal antibody titers and of all isotypes. Fusion of B cells from these mice with SP2/O-BirA cells generated high-affinity antibodies (KACAbs) which were directly and site-specifically biotinylated. We have successfully used this method to generate a range of biotinylated monoclonal antibodies for a large variety of cellular and viral antigens. Thus, we present the KAC/SP2-BirA system as a new standard for the generation of site-specific biotinylated monoclonal antibodies.

KAC mutation; biotin; Ig kappa; BirA; monoclonal antibodies

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