Phage Display Analysis of Monoclonal Antibody Binding to Anthrax Toxin Lethal Factor

Goldstein J, et al., 9(7), 221, Toxins, 2017

Anthrax is caused by a gram-positive bacterium called Bacillus anthracis. AVR1674 and AVR1675 are murine monoclonal antibodies (mAbs) that are highly specific to anthrax toxin lethal factor (LF) and lethal toxin (LTx). These mAbs have been used for the development of mass spectrometry-based anthrax toxin detection tests. Reported herein is the ability of AVR1674 and AVR1675 mAbs to improve LF cleavage of specific peptide substrates in vitro, their remarkable similarity in sequence and functional activity. A panel of anti-LF mAbs binding to rLF, competitive binding of mAbs (AVR1674 and AVR1675) to rLF, and binding kinetics of AVR1675 mAb-peptide interactions were assessed using Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED system equipped with Streptavidin (SA) Biosensor probes was used to perform all BLI assays. The binding of anti-LF mAbs to rLF was studied by immobilizing biotinylated rLF onto SA sensor tips. Association kinetics were studied by dipping the biotinylated rLF loaded sensor tips in a concentration series of purified mAb IgG solutions. Inorder to determine the competition for available LF binding sites, sensor tips with preformed LF-IgG complex was immersed in AVR1675. To study mAb-peptide interactions, SA sensor tips were loaded with biotinylated synthetic oligopeptides. Subsequently, oligopeptide loaded sensor tips were immersed in various concentrations of purified mAb (AVR1675) solutions. The Octet data were fitted globally to a 1:1 Langmuir binding model. The kinetic rate constants (kon and koff) and the binding constant, KD (KD = kon/koff) were determined for all interactions. Overall findings of this study is expected to contribute significantly to future development of function-based LF detection assays with enhanced analytical sensitivity.

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