Production and Isolation of Immunoglobulin G (IgG) Against Antibiotic-Resistant Bacteria for Immunological Treatment"

The global health threat due to the emergence of microorganisms resistant to multiple types of antibiotics continues to escalate, compounding the burden of life during the ongoing COVID-19 pandemic. It is estimated that antimicrobial resistance (AMR) causes more than 700,000 deaths per year, and this figure is projected to rise to approximately 10 million deaths annually by 2050, making it the leading cause of death. In response, major global organizations such as the OIE, WHO, and FAO have adopted the One Health approach to address this challenge. Various efforts have been made, primarily aimed at controlling the rapid acceleration of AMR, as the discovery of reliable new antibiotics generally lags behind the speed at which AMR—especially multi-drug resistance (MDR)—emerges. This study aims to produce anti-AMR IgG antibodies using multi-drug-resistant Escherichia coli (E. coli) as a model for immunotherapy, similar to the use of anti-tetanus serum (ATS), anti-rabies serum (ARS), and anti-venom serum in clinical treatment. Six isolates of E. coli, which have been characterized and found to carry plasmids conferring resistance to eight different antibiotics (hereafter referred to as resistant E. coli), will be cultured in broth media. The harvested cell pellets will be lysed and processed into a bacterin vaccine using both Complete and Incomplete Freund’s Adjuvant. This vaccine will then be administered in vivo to experimental horses following the standard procedures for hyperimmune serum production. Sero-conversion, indicating the production of hyperimmune serum in the horses, will be evaluated in vitro using ELISA and/or Western Blotting to assess homologous reactivity between the resistant E. coli and the hyperimmune equine serum. The specific IgG-antigen interactions will be confirmed using goat anti-horse conjugates labeled with HRP or alkaline phosphatase. Subsequently, the IgG antibodies will be purified from the horse serum using Sepharose gel column chromatography combined with Protein A affinity separation. The neutralization capacity of the purified IgG against the resistant E. coli will be tested in vitro using agar plate assays. The absence of resistant E. coli growth on solid media—compared to the continued growth of control E. coli—will confirm the successful production and isolation of functional anti-resistant E. coli IgG antibodies. These antibodies could potentially be developed further for preclinical trials in animal models, and eventually for clinical therapeutic trials in humans