Production and Characterization of F(Ab’)2 Fragments Obtained by Enzymatic Digestion from Murine Anti-MRSA PBP2a Monoclonal Antibodies

Abstract

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a worldwide health problem. In a previous study, a murine monoclonal antibody (mMAB), capable of binding to PBP2a within MRSA strains, was generated. F(ab') antibody fragments are widely described in the literature as immunochemical tools and reagents for diagnostics and therapeutics, particularly because of their low immunogenicity and rapid pharmacokinetics. In this study, F(ab') fragments from mMAB were generated by enzymatic digestion, using pepsin. They were purified by affinity chromatography using protein A and concentrated by a MWCO 50 kDa filtration unit. The results indicate that it is possible to obtain F(ab') fragments by pepsin digestion. ELISA, western blotting, and fluorescence microscopy data demonstrated that F(ab') affinity for PBP2a is not lost even after the enzymatic digestion process. As expected, in the pharmacokinetics tests, F(ab') presented a faster elimination (between 12 and 18 h) compared to IgG. These F(ab') fragments could be used in future immunodiagnostic applications, including in vitro or in situ radiolabeling and in the treatment of infections caused by this important pathogen.

Human polyspecific immunoglobulin attenuates group A streptococcal virulence factor activity and reduces disease severity in a murine necrotizing fasciitis model

“…We could show that in vitro F(ab 0 )2 fragments inhibited SLO and Sda1 activity comparably to IVIG. Nevertheless, in vivo experiments are required in order to assess possible drawbacks, such as faster degradation and elimination of the smaller F(ab 0 )2 fragments compared to full-length IVIG [27,28].…”

Monoclonal antibody anti-PBP2a protects mice against MRSA (methicillin-resistant Staphylococcus aureus) infections

“…We recently reported the generation and characterization of murine MAbs that specifically bind PBP2a in vitro with high affinity [13]. In another study, we demonstrated that this anti-PBP2a antibody recognizes PBP2a protein on the bacterial surface using an immunofluorescence assay [14]. Here, we demonstrate that this MAb also confers protection in a murine model administered both prophylactic and therapeutic treatments.…”

“…aureus [11], confirming the specificity of anti-PBP2a antibodies against MRSA. The MAb presented here was derived from this work, and it was obtained and characterized in previous studies [11,13,14]. We previously demonstrated that this MAb binds strongly to recombinant PBP2a and recognizes native PBP2a protein on the bacteria.…”

Antibody-Based Agents in the Management of Antibiotic-Resistant Staphylococcus aureus Diseases

“…Results obtained by Naghshbandi et al showed that passive immunization with rabbit purified polyclonal antibodies against recombinant PBP2a results in a significant increase in the survival rate of mice challenged with a lethal dose of MRSA and reduction of bacterial spread to the internal organs, confirming the potential importance of passive immunization with anti-PBP2a antibodies in controlling MRSA infections [ 81 ]. Monoclonal antibodies against PBP2a have also been generated and extensively characterized, but their efficacy in immunoprophylaxisis is still to be determined [ 82 , 83 ].…”