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Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

View Article: PubMed Central - PubMed

ABSTRACT

Tetanus neurotoxin (TeNT) produced by Clostridiumtetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

No MeSH data available.


ELISA characterization of a partial phage-scFv clone binding to different proteins. Assays were performed by immobilizing TeNT-Hc, F1, V, IFN-ω, and BSA-coated on a polystyrene plate. Phage-scFvs derived from the library that were reactive with the coated antigen, were detected with a 1:5000 dilution of horseradish peroxidase (HRP)–conjugated anti-M13 antibody. The results of the assay are shown as the absorbance at 492 nm/630 nm. Assays were performed in triplicate, and the range is shown.
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toxins-08-00266-f001: ELISA characterization of a partial phage-scFv clone binding to different proteins. Assays were performed by immobilizing TeNT-Hc, F1, V, IFN-ω, and BSA-coated on a polystyrene plate. Phage-scFvs derived from the library that were reactive with the coated antigen, were detected with a 1:5000 dilution of horseradish peroxidase (HRP)–conjugated anti-M13 antibody. The results of the assay are shown as the absorbance at 492 nm/630 nm. Assays were performed in triplicate, and the range is shown.

Mentions: Approximately 85% of the clones could specially bind to TeNT-Hc, with no cross-reactivity to other proteins (Figure 1). A total of 12 clones were selected based on the high OD values (OD > 1.5) that were sent for sequencing analysis. Eight different sequences were identified from the 12 clones that were sequenced.


Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library
ELISA characterization of a partial phage-scFv clone binding to different proteins. Assays were performed by immobilizing TeNT-Hc, F1, V, IFN-ω, and BSA-coated on a polystyrene plate. Phage-scFvs derived from the library that were reactive with the coated antigen, were detected with a 1:5000 dilution of horseradish peroxidase (HRP)–conjugated anti-M13 antibody. The results of the assay are shown as the absorbance at 492 nm/630 nm. Assays were performed in triplicate, and the range is shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037492&req=5

toxins-08-00266-f001: ELISA characterization of a partial phage-scFv clone binding to different proteins. Assays were performed by immobilizing TeNT-Hc, F1, V, IFN-ω, and BSA-coated on a polystyrene plate. Phage-scFvs derived from the library that were reactive with the coated antigen, were detected with a 1:5000 dilution of horseradish peroxidase (HRP)–conjugated anti-M13 antibody. The results of the assay are shown as the absorbance at 492 nm/630 nm. Assays were performed in triplicate, and the range is shown.
Mentions: Approximately 85% of the clones could specially bind to TeNT-Hc, with no cross-reactivity to other proteins (Figure 1). A total of 12 clones were selected based on the high OD values (OD > 1.5) that were sent for sequencing analysis. Eight different sequences were identified from the 12 clones that were sequenced.

View Article: PubMed Central - PubMed

ABSTRACT

Tetanus neurotoxin (TeNT) produced by Clostridiumtetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

No MeSH data available.