Phage Display Analysis of Monoclonal Antibody Binding to Anthrax Toxin Lethal Factor
Jason M. Goldstein,
Joo Lee,
Xiaoling Tang,
Anne E. Boyer,
John R. Barr,
Dennis A. Bagarozzi Jr.,
Conrad P. Quinn
Affiliations
Jason M. Goldstein
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
Joo Lee
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
Xiaoling Tang
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
Anne E. Boyer
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
John R. Barr
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
Dennis A. Bagarozzi Jr.
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
Conrad P. Quinn
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
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.
