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Interaction between Shiga toxin and monoclonal antibodies: binding characteristics and in vitro neutralizing abilities.

Rocha LB, Luz DE, Moraes CT, Caravelli A, Fernandes I, Guth BE, Horton DS, Piazza RM - Toxins (Basel) (2012)

Bottom Line: In contrast, 2E11 recognized the A subunit of Stx2, was stable up to 70 ºC, had a high dissociation constant of 6.1 × 10(-10) M, and detected as little as 12.5 ng of Stx2.These MAb amounts reversed 25 to 80% of the cytotoxicity triggered by different STEC isolates.In conclusion, these MAbs show suitable characteristics for their use in STEC diagnosis and encourage future studies to investigate their protective efficacy.

View Article: PubMed Central - PubMed

Affiliation: Bacteriology Laboratory, Butantan Institute, São Paulo, SP, Brazil. leticiarocha@butantan.gov.br

ABSTRACT
Monoclonal antibodies (MAbs) have been employed either for diagnosis or treatment of infections caused by different pathogens. Specifically for Shiga toxin-producing Escherichia coli (STEC), numerous immunoassays have been developed for STEC diagnosis, showing variability in sensitivity and specificity when evaluated by reference laboratories, and no therapy or vaccines are currently approved. Thus, the aim of this work was the characterization of the interaction between MAbs against Stx1 and Stx2 toxins and their neutralizing abilities to enable their use as tools for diagnosis and therapy. The selected clones designated 3E2 (anti-Stx1) and 2E11 (anti-Stx2) were classified as IgG1. 3E2 recognized the B subunit of Stx1 with an affinity constant of 2.5 × 10(-10) M, detected as little as 6.2 ng of Stx1 and was stable up to 50 ºC. In contrast, 2E11 recognized the A subunit of Stx2, was stable up to 70 ºC, had a high dissociation constant of 6.1 × 10(-10) M, and detected as little as 12.5 ng of Stx2. Neutralization tests showed that 160 ng of 3E2 MAb inhibited 80% of Stx1 activity and 500 µg 2E11 MAb were required for 60% inhibition of Stx2 activity. These MAb amounts reversed 25 to 80% of the cytotoxicity triggered by different STEC isolates. In conclusion, these MAbs show suitable characteristics for their use in STEC diagnosis and encourage future studies to investigate their protective efficacy.

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Related in: MedlinePlus

A and B—anti-Stx1 MAb showed cross-reactivity and was able to inhibit between 70 and 80% of both toxins ((A) Stx1 and (B) Stx2). Higher concentrations of anti-Stx2 MAb (100 to 500 µg) were necessary to neutralize Stx2 activity (C), but not Stx1 (data not shown). Bars represent the mean and the standard errors of the percentage of duplicates of three independent experiments.
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toxins-04-00729-f004: A and B—anti-Stx1 MAb showed cross-reactivity and was able to inhibit between 70 and 80% of both toxins ((A) Stx1 and (B) Stx2). Higher concentrations of anti-Stx2 MAb (100 to 500 µg) were necessary to neutralize Stx2 activity (C), but not Stx1 (data not shown). Bars represent the mean and the standard errors of the percentage of duplicates of three independent experiments.

Mentions: The CD50 of each toxin was determined prior to neutralization assays using anti-Stx1 and anti-Stx2 MAbs. CD50 was defined as 10 ng and 500 ng for Stx1 and Stx2, respectively. Concentrations of anti-Stx1 MAb ranging from 1.28 ng to 100 µg neutralized between 30 and 80% of Stx1 cytotoxic activity (Figure 4A). Anti-Stx1 MAb showed cross-reactivity (Figure 4B), i.e., it inhibited both Stx1 and Stx2 activity to the same extent. On the other hand, 100 µg or 200 µg anti-Stx2 MAb were necessary to neutralize ca. 35% of Stx2 activity, and 60% of neutralization was only achieved using 500 µg MAb (Figure 4C). No apparent cell damage was observed in any MAb concentration.


Interaction between Shiga toxin and monoclonal antibodies: binding characteristics and in vitro neutralizing abilities.

Rocha LB, Luz DE, Moraes CT, Caravelli A, Fernandes I, Guth BE, Horton DS, Piazza RM - Toxins (Basel) (2012)

A and B—anti-Stx1 MAb showed cross-reactivity and was able to inhibit between 70 and 80% of both toxins ((A) Stx1 and (B) Stx2). Higher concentrations of anti-Stx2 MAb (100 to 500 µg) were necessary to neutralize Stx2 activity (C), but not Stx1 (data not shown). Bars represent the mean and the standard errors of the percentage of duplicates of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475226&req=5

toxins-04-00729-f004: A and B—anti-Stx1 MAb showed cross-reactivity and was able to inhibit between 70 and 80% of both toxins ((A) Stx1 and (B) Stx2). Higher concentrations of anti-Stx2 MAb (100 to 500 µg) were necessary to neutralize Stx2 activity (C), but not Stx1 (data not shown). Bars represent the mean and the standard errors of the percentage of duplicates of three independent experiments.
Mentions: The CD50 of each toxin was determined prior to neutralization assays using anti-Stx1 and anti-Stx2 MAbs. CD50 was defined as 10 ng and 500 ng for Stx1 and Stx2, respectively. Concentrations of anti-Stx1 MAb ranging from 1.28 ng to 100 µg neutralized between 30 and 80% of Stx1 cytotoxic activity (Figure 4A). Anti-Stx1 MAb showed cross-reactivity (Figure 4B), i.e., it inhibited both Stx1 and Stx2 activity to the same extent. On the other hand, 100 µg or 200 µg anti-Stx2 MAb were necessary to neutralize ca. 35% of Stx2 activity, and 60% of neutralization was only achieved using 500 µg MAb (Figure 4C). No apparent cell damage was observed in any MAb concentration.

Bottom Line: In contrast, 2E11 recognized the A subunit of Stx2, was stable up to 70 ºC, had a high dissociation constant of 6.1 × 10(-10) M, and detected as little as 12.5 ng of Stx2.These MAb amounts reversed 25 to 80% of the cytotoxicity triggered by different STEC isolates.In conclusion, these MAbs show suitable characteristics for their use in STEC diagnosis and encourage future studies to investigate their protective efficacy.

View Article: PubMed Central - PubMed

Affiliation: Bacteriology Laboratory, Butantan Institute, São Paulo, SP, Brazil. leticiarocha@butantan.gov.br

ABSTRACT
Monoclonal antibodies (MAbs) have been employed either for diagnosis or treatment of infections caused by different pathogens. Specifically for Shiga toxin-producing Escherichia coli (STEC), numerous immunoassays have been developed for STEC diagnosis, showing variability in sensitivity and specificity when evaluated by reference laboratories, and no therapy or vaccines are currently approved. Thus, the aim of this work was the characterization of the interaction between MAbs against Stx1 and Stx2 toxins and their neutralizing abilities to enable their use as tools for diagnosis and therapy. The selected clones designated 3E2 (anti-Stx1) and 2E11 (anti-Stx2) were classified as IgG1. 3E2 recognized the B subunit of Stx1 with an affinity constant of 2.5 × 10(-10) M, detected as little as 6.2 ng of Stx1 and was stable up to 50 ºC. In contrast, 2E11 recognized the A subunit of Stx2, was stable up to 70 ºC, had a high dissociation constant of 6.1 × 10(-10) M, and detected as little as 12.5 ng of Stx2. Neutralization tests showed that 160 ng of 3E2 MAb inhibited 80% of Stx1 activity and 500 µg 2E11 MAb were required for 60% inhibition of Stx2 activity. These MAb amounts reversed 25 to 80% of the cytotoxicity triggered by different STEC isolates. In conclusion, these MAbs show suitable characteristics for their use in STEC diagnosis and encourage future studies to investigate their protective efficacy.

Show MeSH
Related in: MedlinePlus