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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.


Related in: MedlinePlus

The biological activity of 2-7G, 2-2D, or S-4-7H, either individually or in combination, was determined using a tetanus toxin neutralization assay in mice. First, 20 µg total antibodies was mixed with 20 mouse LD50s of toxin and injected intraperitoneally (i.p.). Then, the time-to-death and the number of surviving mice were determined. No single mAb exhibited a significant protection against 20 LD50s, but could partly protect or prolong the time-to-death. All mice survived the challenge with 20 LD50s when administered the mixture of 2-7G, 2-2D, and S-4-7H IgG4.
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toxins-08-00266-f007: The biological activity of 2-7G, 2-2D, or S-4-7H, either individually or in combination, was determined using a tetanus toxin neutralization assay in mice. First, 20 µg total antibodies was mixed with 20 mouse LD50s of toxin and injected intraperitoneally (i.p.). Then, the time-to-death and the number of surviving mice were determined. No single mAb exhibited a significant protection against 20 LD50s, but could partly protect or prolong the time-to-death. All mice survived the challenge with 20 LD50s when administered the mixture of 2-7G, 2-2D, and S-4-7H IgG4.

Mentions: A mouse assay was used to detect the toxin neutralization of 2-7G, 2-2D, and S-4-7H. Using the horse polyclonal anti-TeNT-Hc antibodies as a positive control, 20 µg of single antibody or antibody mixture was premixed with 20 LD50s TeNT and injected i.p. into the mice. The mice challenged with 20 LD50s TeNT all died within 24 h. Although no single antibody exhibited significant protection against 20 LD50s, 2-7G could protect 70% mice from death, and 2-2D or S-4-7H could prolong the time to death. All mice survived the challenge with 20 LD50s when administered the mixture of 2-7G, 2-2D, and S-4-7H (Figure 7), and the combination of the two antibodies could protect 70%–100% mice from death. The results indicated that 2-7G, 2-2D, and S-4-7H had a certain degree of protection against TeNT, whereas the combination of two or three antibodies had a greater potency of in vivo toxin neutralization. This may be due to the increase in the blockade of the toxin surface that binds to cellular receptors.


Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library
The biological activity of 2-7G, 2-2D, or S-4-7H, either individually or in combination, was determined using a tetanus toxin neutralization assay in mice. First, 20 µg total antibodies was mixed with 20 mouse LD50s of toxin and injected intraperitoneally (i.p.). Then, the time-to-death and the number of surviving mice were determined. No single mAb exhibited a significant protection against 20 LD50s, but could partly protect or prolong the time-to-death. All mice survived the challenge with 20 LD50s when administered the mixture of 2-7G, 2-2D, and S-4-7H IgG4.
© Copyright Policy
Related In: Results  -  Collection

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

toxins-08-00266-f007: The biological activity of 2-7G, 2-2D, or S-4-7H, either individually or in combination, was determined using a tetanus toxin neutralization assay in mice. First, 20 µg total antibodies was mixed with 20 mouse LD50s of toxin and injected intraperitoneally (i.p.). Then, the time-to-death and the number of surviving mice were determined. No single mAb exhibited a significant protection against 20 LD50s, but could partly protect or prolong the time-to-death. All mice survived the challenge with 20 LD50s when administered the mixture of 2-7G, 2-2D, and S-4-7H IgG4.
Mentions: A mouse assay was used to detect the toxin neutralization of 2-7G, 2-2D, and S-4-7H. Using the horse polyclonal anti-TeNT-Hc antibodies as a positive control, 20 µg of single antibody or antibody mixture was premixed with 20 LD50s TeNT and injected i.p. into the mice. The mice challenged with 20 LD50s TeNT all died within 24 h. Although no single antibody exhibited significant protection against 20 LD50s, 2-7G could protect 70% mice from death, and 2-2D or S-4-7H could prolong the time to death. All mice survived the challenge with 20 LD50s when administered the mixture of 2-7G, 2-2D, and S-4-7H (Figure 7), and the combination of the two antibodies could protect 70%–100% mice from death. The results indicated that 2-7G, 2-2D, and S-4-7H had a certain degree of protection against TeNT, whereas the combination of two or three antibodies had a greater potency of in vivo toxin neutralization. This may be due to the increase in the blockade of the toxin surface that binds to cellular receptors.

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.


Related in: MedlinePlus