RESUMO
Immunoglobulin purification from different biological fluids is considered one of the most critical steps in antibody production for diagnostic, therapeutic, and research purposes. The current study aimed to elucidate the role of the different aryl substituents in triazine-based affinity ligands on the performance of an affinity chromatography purification media to separate immunoglobulin G (IgG). The biomimetic triazine-based affinity ligand was chosen as a varied containing fix spacer and support. The sepharose beads were activated by epichlorohydrin, and five types of aryl substituents were replaced in the triazine ring and covalently immobilized to the resin surface by 1, 4-diaminobutane spacer. All affinity resins with various ligands were characterized and validated using FTIR, SEM, EDX, and microscopic images. The findings revealed that using R1=3-aminophenol and R2=3-aminophenol substituents in the triazine ring, as affinity ligands attached to the sepharose surface with a 10-atom linker CAES-6B-Cl@R1= MAF, R2= MAF (No. 4), leads to better purification of IgG from human and rabbit plasma with 22.8 mg/mL resin binding capacity in 73±5% yield and 95% of purity. All results confirmed that the designed triazine-based affinity ligands could effectively purify IgG compatible with a fast and low-cost approach.
Assuntos
Imunoglobulina G , Triazinas , Animais , Humanos , Coelhos , Ligantes , Imunoglobulina G/metabolismo , Triazinas/química , Sefarose/química , Biomimética , Cromatografia de Afinidade/métodosRESUMO
BACKGROUND: Pseudomonas aeruginosa is the leading cause of nosocomial infections, especially in people with a compromised immune system. Targeting virulence factors by neutralizing antibodies is a novel paradigm for the treatment of antibiotic-resistant pseudomonas infections. In this respect, exotoxin A is one of the most potent virulence factors in P. aeruginosa. The present study was carried out to identify a novel human scFv antibody against the P. aeruginosa exotoxin A domain I (ExoA-DI) from a human scFv phage library. METHODS: The recombinant ExoA-DI of P. aeruginosa was expressed in E. coli, purified by Ni-NTA column, and used for screening of human antibody phage library. A novel screening procedure was conducted to prevent the elimination of rare specific clones. The phage clone with high reactivity was evaluated by ELISA and western blot. RESULTS: Based on the results of polyclonal phage ELISA, the fifth round of biopanning leads to the isolation of several ExoA-DI reactive clones. One positive clone with high affinity was selected by monoclonal phage ELISA and used for antibody expression. The purified scFv showed high reactivity with the recombinant domain I and full-length native exotoxin A. CONCLUSIONS: The purified anti-exotoxin A scFv displayed high specificity against exotoxin A. The human scFv identified in this study could be the groundwork for developing a novel therapeutic agent to control P. aeruginosa infections.
Assuntos
ADP Ribose Transferases/imunologia , Toxinas Bacterianas/imunologia , Exotoxinas/imunologia , Pseudomonas aeruginosa/imunologia , Anticorpos de Cadeia Única/imunologia , Fatores de Virulência/imunologia , ADP Ribose Transferases/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Toxinas Bacterianas/genética , Escherichia coli/genética , Exotoxinas/genética , Humanos , Biblioteca de Peptídeos , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/isolamento & purificação , Fatores de Virulência/genética , Exotoxina A de Pseudomonas aeruginosaRESUMO
Escalating antibiotic resistance is now a serious menace to global public health. It may be led to the emergence of "postantibiotic age" in which most of infections are untreatable. At present, there is an essential need to explore novel therapeutic strategies as a strong and sustainable pipeline to combat antibiotic-resistant infections. This review focuses on recent advances in this area including therapeutic antibodies, antimicrobial peptides, vaccines, gene therapy, genome editing, and phage therapy for tackling drug-resistant infections.