Phage Display Analysis of Monoclonal Antibody Binding to Anthrax Toxin Lethal Factor
Reagent and Diagnostic Services Branch, Division of Scientific Resources, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, MS-A03, 1600 Clifton Road, Atlanta, GA 30333, USA
Clinical Chemistry Branch, Division of Laboratory Services, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Hwy, NE, Atlanta, GA 30341, USA
Meningitis and Vaccine Preventable Diseases Branch, Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, MS-D17, 1600 Clifton Road, Atlanta, GA 30333, USA
Author to whom correspondence should be addressed.
Academic Editor: Shihui Liu
Toxins 2017, 9(7), 221; https://doi.org/10.3390/toxins9070221
Received: 27 April 2017 / Revised: 28 June 2017 / Accepted: 10 July 2017 / Published: 13 July 2017
(This article belongs to the Collection Anthrax Toxins)
AVR1674 and AVR1675 are monoclonal antibodies (mAbs) that bind with high specificity to anthrax toxin lethal factor (LF) and lethal toxin (LTx). These mAbs have been used as pivotal reagents to develop anthrax toxin detection tests using mass spectrometry. The mAbs were demonstrated to bind LF with good affinity (KD 10−7–10−9 M) and to enhance LF-mediated cleavage of synthetic peptide substrates in vitro. Sequence analysis indicated that the mAbs shared 100% amino acid identity in their complementarity determining regions (CDR). A phage display library based on a combinatorial library of random heptapeptides fused to the pIII coat protein of M13 phage was enriched and screened to identify peptide sequences with mAb binding properties. Selection and sequence analysis of 18 anti-LF-reactive phage clones identified a 7-residue (P1–P7) AVR1674/1675 consensus target binding sequence of TP1-XP2-K/RP3-DP4-D/EP5-ZP6-X/ZP7 (X = aromatic, Z = non-polar). The phage peptide sequence with highest affinity binding to AVR1674/1675 was identified as T-F-K-D-E-I-V. Synthetic oligopeptides were designed based on the phage sequences and interacted with mAbs with high affinity (KD ~ 10−9 M). Single amino acid substitutions of A, H, or Q in the peptides identified positions P1–P5 as critical residues for mAb-peptide interactions. CLUSTALW alignment of phage sequences with native LF implicated residues 644–650 (sequence T-H-Q-D-E-I-Y) as a putative linear epitope component located within a structural loop (L2) of LF Domain IV. The activation effects of these mAbs contribute to the analytic sensitivity of function-based LF detection assays.