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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

Vero cell cytotoxicity assay (VCA) and neutralization assays with culture supernatant of 46 shiga toxin-producing Escherichia coli (STEC) isolates producing Stx1 and/or Stx2 incubated with (blue) or without (red) anti-Stx1 and anti-Stx2 MAbs followed by cell incubation. Cytotoxicity and neutralization were determined after staining the cells with crystal violet and measuring absorbance at 595 nm. Means and variances were significantly different (p < 0.0001) by Student’s t-test and 2-way ANOVA comparing cytotoxicity and neutralization groups. Bars represent the OD means and standard errors median of duplicates of three independent experiments. A high OD value means high cell viability.
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toxins-04-00729-f005: Vero cell cytotoxicity assay (VCA) and neutralization assays with culture supernatant of 46 shiga toxin-producing Escherichia coli (STEC) isolates producing Stx1 and/or Stx2 incubated with (blue) or without (red) anti-Stx1 and anti-Stx2 MAbs followed by cell incubation. Cytotoxicity and neutralization were determined after staining the cells with crystal violet and measuring absorbance at 595 nm. Means and variances were significantly different (p < 0.0001) by Student’s t-test and 2-way ANOVA comparing cytotoxicity and neutralization groups. Bars represent the OD means and standard errors median of duplicates of three independent experiments. A high OD value means high cell viability.

Mentions: After determining the neutralization point for both MAbs, their ability to neutralize the cytotoxic activity of the STEC strains, belonging to several serotypes and showing either stx1, stx2 or stx1/stx2 genes, was investigated. We observed that Stx activity was neutralized (from 25 to 80%) by the MAbs in all isolates (Figure 5). Besides, using both MAbs, we were able to neutralize Stx1 and Stx2 expressed by isolates. Cellular integrity was maintained at these MAb concentrations in the absence of toxins. Means and variances were significantly different (p < 0.0001) by Student’s t-test and 2-way ANOVA, comparing the cytotoxicity and neutralization groups (Figure 5).


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)

Vero cell cytotoxicity assay (VCA) and neutralization assays with culture supernatant of 46 shiga toxin-producing Escherichia coli (STEC) isolates producing Stx1 and/or Stx2 incubated with (blue) or without (red) anti-Stx1 and anti-Stx2 MAbs followed by cell incubation. Cytotoxicity and neutralization were determined after staining the cells with crystal violet and measuring absorbance at 595 nm. Means and variances were significantly different (p < 0.0001) by Student’s t-test and 2-way ANOVA comparing cytotoxicity and neutralization groups. Bars represent the OD means and standard errors median of duplicates of three independent experiments. A high OD value means high cell viability.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

toxins-04-00729-f005: Vero cell cytotoxicity assay (VCA) and neutralization assays with culture supernatant of 46 shiga toxin-producing Escherichia coli (STEC) isolates producing Stx1 and/or Stx2 incubated with (blue) or without (red) anti-Stx1 and anti-Stx2 MAbs followed by cell incubation. Cytotoxicity and neutralization were determined after staining the cells with crystal violet and measuring absorbance at 595 nm. Means and variances were significantly different (p < 0.0001) by Student’s t-test and 2-way ANOVA comparing cytotoxicity and neutralization groups. Bars represent the OD means and standard errors median of duplicates of three independent experiments. A high OD value means high cell viability.
Mentions: After determining the neutralization point for both MAbs, their ability to neutralize the cytotoxic activity of the STEC strains, belonging to several serotypes and showing either stx1, stx2 or stx1/stx2 genes, was investigated. We observed that Stx activity was neutralized (from 25 to 80%) by the MAbs in all isolates (Figure 5). Besides, using both MAbs, we were able to neutralize Stx1 and Stx2 expressed by isolates. Cellular integrity was maintained at these MAb concentrations in the absence of toxins. Means and variances were significantly different (p < 0.0001) by Student’s t-test and 2-way ANOVA, comparing the cytotoxicity and neutralization groups (Figure 5).

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