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Epitope characterization and variable region sequence of f1-40, a high-affinity monoclonal antibody to botulinum neurotoxin type a (Hall strain).

Scotcher MC, McGarvey JA, Johnson EA, Stanker LH - PLoS ONE (2009)

Bottom Line: F1-40 was found to bind a peptide fragment of BoNT/A, designated L1-3, which spans from T125 to L200.The epitope of F1-40 was localized to a single exposed loop (ss4, ss5) on the Lc of BoNT.Furthermore amino acids Q138, P139 and D140 forming the tip of the loop appear critical for binding.

View Article: PubMed Central - PubMed

Affiliation: United States Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Albany, California, United States of America.

ABSTRACT

Background: Botulism, an often fatal neuroparalytic disease, is caused by botulinum neurotoxins (BoNT) which consist of a family of seven serotypes (A-H) produced by the anaerobic bacterium Clostridium botulinum. BoNT, considered the most potent biological toxin known, is a 150 kDa protein consisting of a 100 kDa heavy-chain (Hc) and a 50 kDa light-chain (Lc). F1-40 is a mouse-derived, IgG1 monoclonal antibody that binds the light chain of BoNT serotype A (BoNT/A) and is used in a sensitive immunoassay for toxin detection. We report the fine epitope mapping of F1-40 and the deduced amino acid sequence of the variable regions of the heavy and light chains of the antibody.

Methods and findings: To characterize the binding epitope of F1-40, three complementary experimental approaches were selected. Firstly, recombinant peptide fragments of BoNT/A light-chain were used in Western blots to identify the epitope domains. Secondly, a peptide phage-display library was used to identify the specific amino acid sequences. Thirdly, the three-dimensional structure of BoNT/A was examined in silico, and the amino acid sequences determined from the phage-display studies were mapped onto the three-dimensional structure in order to visualize the epitope. F1-40 was found to bind a peptide fragment of BoNT/A, designated L1-3, which spans from T125 to L200. The motif QPDRS was identified by phage-display, and was mapped to a region within L1-3. When the three amino acids Q138, P139 and D140 were all mutated to glycine, binding of F1-40 to the recombinant BoNT/A light chain peptide was abolished. Q-138, P-139 and D-140 form a loop on the external surface of BoNT/A, exposed to solvent and accessible to F1-40 binding.

Conclusions: The epitope of F1-40 was localized to a single exposed loop (ss4, ss5) on the Lc of BoNT. Furthermore amino acids Q138, P139 and D140 forming the tip of the loop appear critical for binding.

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Competition ELISA for F1-40 by Lc peptide fragment mutants in solution.The percent inhibition of binding of mAb F1-40 to immobilized Lc peptide fragment on a 96-well plate by various peptide fragments in solution (♦, Lc; ▵ Lc-Δ; ○ Lc-QPD; ▪ Lc-RS) was calculated by the formula (1-B/B0)×100, where B = luminescent counts at each concentration of fusion peptide in solution, and B0 = luminescent counts at 0 µg/mL fusion peptide in solution. Data is presented±1 standard error, n = 4.
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pone-0004924-g005: Competition ELISA for F1-40 by Lc peptide fragment mutants in solution.The percent inhibition of binding of mAb F1-40 to immobilized Lc peptide fragment on a 96-well plate by various peptide fragments in solution (♦, Lc; ▵ Lc-Δ; ○ Lc-QPD; ▪ Lc-RS) was calculated by the formula (1-B/B0)×100, where B = luminescent counts at each concentration of fusion peptide in solution, and B0 = luminescent counts at 0 µg/mL fusion peptide in solution. Data is presented±1 standard error, n = 4.

Mentions: Competition ELISA assays were performed to better quantify the differences of antibody binding to the mutant peptides observed in the above Western blotting experiments. Results from these experiments are shown in Figure 5. Peptide Lc was the most effective at competing binding of F1-40 to immobilized Lc, with 50% inhibition achieved at a concentration of ∼3.5 µg/mL. Mutant peptide Lc-RS was the next most effective competitor resulting in 50% inhibition of control activity at ∼24 µg/mL, roughly 7-fold higher than that observed for the recombinant Lc peptide. Finally, mutants Lc-Δ and Lc-QPD did not compete for F1-40 binding under these conditions, even at the highest concentration of peptide used (60 µg/mL).


Epitope characterization and variable region sequence of f1-40, a high-affinity monoclonal antibody to botulinum neurotoxin type a (Hall strain).

Scotcher MC, McGarvey JA, Johnson EA, Stanker LH - PLoS ONE (2009)

Competition ELISA for F1-40 by Lc peptide fragment mutants in solution.The percent inhibition of binding of mAb F1-40 to immobilized Lc peptide fragment on a 96-well plate by various peptide fragments in solution (♦, Lc; ▵ Lc-Δ; ○ Lc-QPD; ▪ Lc-RS) was calculated by the formula (1-B/B0)×100, where B = luminescent counts at each concentration of fusion peptide in solution, and B0 = luminescent counts at 0 µg/mL fusion peptide in solution. Data is presented±1 standard error, n = 4.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654115&req=5

pone-0004924-g005: Competition ELISA for F1-40 by Lc peptide fragment mutants in solution.The percent inhibition of binding of mAb F1-40 to immobilized Lc peptide fragment on a 96-well plate by various peptide fragments in solution (♦, Lc; ▵ Lc-Δ; ○ Lc-QPD; ▪ Lc-RS) was calculated by the formula (1-B/B0)×100, where B = luminescent counts at each concentration of fusion peptide in solution, and B0 = luminescent counts at 0 µg/mL fusion peptide in solution. Data is presented±1 standard error, n = 4.
Mentions: Competition ELISA assays were performed to better quantify the differences of antibody binding to the mutant peptides observed in the above Western blotting experiments. Results from these experiments are shown in Figure 5. Peptide Lc was the most effective at competing binding of F1-40 to immobilized Lc, with 50% inhibition achieved at a concentration of ∼3.5 µg/mL. Mutant peptide Lc-RS was the next most effective competitor resulting in 50% inhibition of control activity at ∼24 µg/mL, roughly 7-fold higher than that observed for the recombinant Lc peptide. Finally, mutants Lc-Δ and Lc-QPD did not compete for F1-40 binding under these conditions, even at the highest concentration of peptide used (60 µg/mL).

Bottom Line: F1-40 was found to bind a peptide fragment of BoNT/A, designated L1-3, which spans from T125 to L200.The epitope of F1-40 was localized to a single exposed loop (ss4, ss5) on the Lc of BoNT.Furthermore amino acids Q138, P139 and D140 forming the tip of the loop appear critical for binding.

View Article: PubMed Central - PubMed

Affiliation: United States Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Albany, California, United States of America.

ABSTRACT

Background: Botulism, an often fatal neuroparalytic disease, is caused by botulinum neurotoxins (BoNT) which consist of a family of seven serotypes (A-H) produced by the anaerobic bacterium Clostridium botulinum. BoNT, considered the most potent biological toxin known, is a 150 kDa protein consisting of a 100 kDa heavy-chain (Hc) and a 50 kDa light-chain (Lc). F1-40 is a mouse-derived, IgG1 monoclonal antibody that binds the light chain of BoNT serotype A (BoNT/A) and is used in a sensitive immunoassay for toxin detection. We report the fine epitope mapping of F1-40 and the deduced amino acid sequence of the variable regions of the heavy and light chains of the antibody.

Methods and findings: To characterize the binding epitope of F1-40, three complementary experimental approaches were selected. Firstly, recombinant peptide fragments of BoNT/A light-chain were used in Western blots to identify the epitope domains. Secondly, a peptide phage-display library was used to identify the specific amino acid sequences. Thirdly, the three-dimensional structure of BoNT/A was examined in silico, and the amino acid sequences determined from the phage-display studies were mapped onto the three-dimensional structure in order to visualize the epitope. F1-40 was found to bind a peptide fragment of BoNT/A, designated L1-3, which spans from T125 to L200. The motif QPDRS was identified by phage-display, and was mapped to a region within L1-3. When the three amino acids Q138, P139 and D140 were all mutated to glycine, binding of F1-40 to the recombinant BoNT/A light chain peptide was abolished. Q-138, P-139 and D-140 form a loop on the external surface of BoNT/A, exposed to solvent and accessible to F1-40 binding.

Conclusions: The epitope of F1-40 was localized to a single exposed loop (ss4, ss5) on the Lc of BoNT. Furthermore amino acids Q138, P139 and D140 forming the tip of the loop appear critical for binding.

Show MeSH
Related in: MedlinePlus