Production of F(ab')₂ from Monoclonal and Polyclonal Antibodies

Antibodies are widely used in therapeutic, diagnostic, and research applications, and antibody derivatives such as F(ab')₂ fragments are used when only a particular antibody region is required. F(ab')₂ can be produced through antibody engineering, but some applications require F(ab')₂ produced from an original formulated antibody or directly from a polyclonal antibody pool. The cysteine protease immunoglobulin-degrading enzyme (IdeS) from Streptococcus pyogenes digests immunoglobulin G (IgG) specifically and efficiently to produce F(ab')₂ . Here we detail the production and purification of recombinant IdeS; its utilization to digest monoclonal or polyclonal antibodies to F(ab')₂ fragments; and F(ab')₂ purification through consecutive affinity chromatography steps. The resultant F(ab')₂ exhibit high purity, retain antigen-binding functionality, and are readily utilizable in various downstream applications. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Production and purification of F(ab')₂ fragments from monoclonal and polyclonal antibodies using IdeS Alternate Protocol: Purification of polyclonal antigen-specific F(ab')₂ fragments from human serum or secretions Support Protocol: Production and purification of IdeS.