Phage Display Analysis of Monoclonal Antibody Binding to Anthrax Toxin Lethal Factor
AbstractAVR1674 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. View Full-Text
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Goldstein, J.M.; Lee, J.; Tang, X.; Boyer, A.E.; Barr, J.R.; Bagarozzi Jr., D.A.; Quinn, C.P. Phage Display Analysis of Monoclonal Antibody Binding to Anthrax Toxin Lethal Factor. Toxins 2017, 9, 221.
Goldstein JM, Lee J, Tang X, Boyer AE, Barr JR, Bagarozzi Jr. DA, Quinn CP. Phage Display Analysis of Monoclonal Antibody Binding to Anthrax Toxin Lethal Factor. Toxins. 2017; 9(7):221.Chicago/Turabian Style
Goldstein, Jason M.; Lee, Joo; Tang, Xiaoling; Boyer, Anne E.; Barr, John R.; Bagarozzi Jr., Dennis A.; Quinn, Conrad P. 2017. "Phage Display Analysis of Monoclonal Antibody Binding to Anthrax Toxin Lethal Factor." Toxins 9, no. 7: 221.