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

Abstract

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 IdeSAlternate Protocol: Purification of polyclonal antigen–specific F(ab′)2 fragments from human serum or secretionsSupport Protocol: Production and purification of IdeS

The impact of IdeS (imlifidase) on allo‐specific, xeno‐reactive, and protective antibodies in a sensitized rhesus macaque model

“…A DNA sequence encoding the Immunoglobulin G‐degrading enzyme of Streptococcus pyogenes (IdeS), Uniprot accession number F8V4V0, 24 was expressed using the pET‐30a (+) vector. The DNA sequence was codon optimized for E. coli expression.…”

“…23 In the present study, we generated IdeS, confirmed in vitro IdeS-mediated rhesus IgG cleavage, and showed in vivo efficacy of IgG with various specificities which are critical in organ transplantation-namely donor-specific (allo), preformed anti-pig (xeno), and CMV-specific (protective) antibodies. F8V4V0, 24 was expressed using the pET-30a (+) vector. The DNA sequence was codon optimized for E. coli expression.…”

A Review on The Clinical Efficacy of Antitetanic Hyperimmune Serum Prepared in Equine ‎Using Freund Adjuvants in Response to Toxoid and Toxin Immunization

“…Consequently, antibodies were documented to offer prophylactic and therapeutic intervention options for disease prevention and control which raise their clinical, diagnostic, and testing applications especially in high-risk populations (Berry, 2018 and Rosenstein et al, 2020).Traditional immunization methods have created an antibody-based humoral response that is highly efficient against contagious diseases that are sensitive to it. Immunization involves introducing an outside material to trigger a certain immune response in the host (Rosenstein et al, 2020).Nowadays, the use of vaccination in the prevention and treatment of autoimmune illnesses, cancer, addictions, allergies, and infectious diseases is extensively being studied. Additionally, vaccination is employed to create physiologically active substances like polyclonal and monoclonal antibodies (Schunk and Macallum, 2005).…”

“…Therefore, the hyperimmune serum as a non-antibiotic biological reagent continues to be developed for research needs, immunodiagnostic tools as well as is considered an alternative treatment to lower the disease recurrence rates. Additionally, the specific antibodies in hyperimmune serum can be used to treat and control diseases in the event of an outbreak (Rosenstein et al, 2020) Considering the numerous applications of hyperimmune serum in research and clinical fields, the preparation method for developing hyperimmune serum against pathogens is essential. There are two methods of hyperimmune serum production, monoclonal antibody production and polyclonal antibody production (Qiu et al, 2016).…”

“…Immunization to tetanus is antibodymediated that can be accomplished with toxoid containing vaccines which stimulate an immune response and production of antitoxin. The currently available antitetanic sera are generally obtained by hyperimmunization of an animal (generally, a horse) by injecting the animal with increasing doses of alum-toxoid (Rosenstein et al, 2020). Injection of antitetanic hyperimmune serum containing antitoxin neutralizes the neurotropic toxin and provides passive immunity in situations when there is a high risk of infection and insufficient time for the body to develop its own immune response, or to reduce the symptoms of ongoing or immunosuppressive diseases such as hypogammaglobulinemia (Lasocka et al, 2021).…”