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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

Immunofluorescence assay after Stx1 or Stx2 interaction with Vero cells for 0, 2, 6 and 24 h. The reaction was carried out by incubating the cells with anti-Stx1 or anti-Stx2 MAbs. Cells labeled with FITC displayed an apple green fluorescence, showing that MAbs were able to recognize the toxins after cell interaction. Reactivity of anti-Stx1 or anti-Stx2 MAbs with cells and the anti-IgG mouse FITC in the absence of toxins was used as the negative control (C-), besides the differential interference contrast (DIC) of negative control. Reactivity was visualized with a confocal laser-scanning microscope (LSM 510 META).
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toxins-04-00729-f003: Immunofluorescence assay after Stx1 or Stx2 interaction with Vero cells for 0, 2, 6 and 24 h. The reaction was carried out by incubating the cells with anti-Stx1 or anti-Stx2 MAbs. Cells labeled with FITC displayed an apple green fluorescence, showing that MAbs were able to recognize the toxins after cell interaction. Reactivity of anti-Stx1 or anti-Stx2 MAbs with cells and the anti-IgG mouse FITC in the absence of toxins was used as the negative control (C-), besides the differential interference contrast (DIC) of negative control. Reactivity was visualized with a confocal laser-scanning microscope (LSM 510 META).

Mentions: The kinetics of the toxins was analyzed by incubating the toxins at 0, 2, 6 and 24 h with Vero cells. After fixation, these interactions were visualized after the MAb reaction (Figure 3). In the early periods (0 and 2 h) each MAb recognized only their respective toxins, but after 6 h both MAbs were able to identify the presence of both toxins in Vero cells, shown by immunofluorescence. Using confocal microscopy, we observed that fluorescence emission with the homologous toxin was more intense. Presence of either Stx1 or Stx2 was observed along the cell when anti-Stx1 was employed (Figure 3). The pattern of recognition by the anti-Stx2 MAbs with either Stx1 or Stx2 was limited to the cell border (Figure 3). Assay specificity was assured by interaction of monoclonal antibodies with Vero cells in the absence of toxin (Figure 3).


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)

Immunofluorescence assay after Stx1 or Stx2 interaction with Vero cells for 0, 2, 6 and 24 h. The reaction was carried out by incubating the cells with anti-Stx1 or anti-Stx2 MAbs. Cells labeled with FITC displayed an apple green fluorescence, showing that MAbs were able to recognize the toxins after cell interaction. Reactivity of anti-Stx1 or anti-Stx2 MAbs with cells and the anti-IgG mouse FITC in the absence of toxins was used as the negative control (C-), besides the differential interference contrast (DIC) of negative control. Reactivity was visualized with a confocal laser-scanning microscope (LSM 510 META).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

toxins-04-00729-f003: Immunofluorescence assay after Stx1 or Stx2 interaction with Vero cells for 0, 2, 6 and 24 h. The reaction was carried out by incubating the cells with anti-Stx1 or anti-Stx2 MAbs. Cells labeled with FITC displayed an apple green fluorescence, showing that MAbs were able to recognize the toxins after cell interaction. Reactivity of anti-Stx1 or anti-Stx2 MAbs with cells and the anti-IgG mouse FITC in the absence of toxins was used as the negative control (C-), besides the differential interference contrast (DIC) of negative control. Reactivity was visualized with a confocal laser-scanning microscope (LSM 510 META).
Mentions: The kinetics of the toxins was analyzed by incubating the toxins at 0, 2, 6 and 24 h with Vero cells. After fixation, these interactions were visualized after the MAb reaction (Figure 3). In the early periods (0 and 2 h) each MAb recognized only their respective toxins, but after 6 h both MAbs were able to identify the presence of both toxins in Vero cells, shown by immunofluorescence. Using confocal microscopy, we observed that fluorescence emission with the homologous toxin was more intense. Presence of either Stx1 or Stx2 was observed along the cell when anti-Stx1 was employed (Figure 3). The pattern of recognition by the anti-Stx2 MAbs with either Stx1 or Stx2 was limited to the cell border (Figure 3). Assay specificity was assured by interaction of monoclonal antibodies with Vero cells in the absence of toxin (Figure 3).

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