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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

Indirect ELISA using MAbs 3E2 and 2E11 and purified toxins Stx1 or Stx2. ELISA microtiter was coated with 0.1 μg of Stx1 (A and B) and Stx2 (C and D). The reaction was carried out using different concentrations of anti-Stx1 MAb, 3E2 (A and C) or anti-Stx2 MAb 2E11 (B and D). The cut-off was defined as 0.1 OD. Cross reactivity occurred at high MAbs concentration.
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toxins-04-00729-f002: Indirect ELISA using MAbs 3E2 and 2E11 and purified toxins Stx1 or Stx2. ELISA microtiter was coated with 0.1 μg of Stx1 (A and B) and Stx2 (C and D). The reaction was carried out using different concentrations of anti-Stx1 MAb, 3E2 (A and C) or anti-Stx2 MAb 2E11 (B and D). The cut-off was defined as 0.1 OD. Cross reactivity occurred at high MAbs concentration.

Mentions: Using 200 ng MAbs to determine their ability to bind to the respective toxin, the detection limit was 6.2 ng for anti-Stx1 and 12.5 ng for anti-Stx2, using an optical density of 0.1 as cut-off (Table 1). The equivalent cut-off was used for determining the reactivity and cross reactivity of MAbs and toxins. Anti-Stx1 (3E2) reacted with 0.1 μg of Stx1 up to 19.5 ng (Figure 2A), 2.5 μg of anti-Stx1 MAb was necessary to detect the same amount of Stx2 (Figure 2B). For anti-Stx2 (2E11) the reactivity with the Stx2 was up to 18.7 pg (Figure 2D) and 0.31 μg was necessary to detect Stx1 (Figure 2C). These results show that cross reactivity occurrs only in the presence of higher MAb concentrations, which are excessive to detected theirs respective toxins.


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)

Indirect ELISA using MAbs 3E2 and 2E11 and purified toxins Stx1 or Stx2. ELISA microtiter was coated with 0.1 μg of Stx1 (A and B) and Stx2 (C and D). The reaction was carried out using different concentrations of anti-Stx1 MAb, 3E2 (A and C) or anti-Stx2 MAb 2E11 (B and D). The cut-off was defined as 0.1 OD. Cross reactivity occurred at high MAbs concentration.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

toxins-04-00729-f002: Indirect ELISA using MAbs 3E2 and 2E11 and purified toxins Stx1 or Stx2. ELISA microtiter was coated with 0.1 μg of Stx1 (A and B) and Stx2 (C and D). The reaction was carried out using different concentrations of anti-Stx1 MAb, 3E2 (A and C) or anti-Stx2 MAb 2E11 (B and D). The cut-off was defined as 0.1 OD. Cross reactivity occurred at high MAbs concentration.
Mentions: Using 200 ng MAbs to determine their ability to bind to the respective toxin, the detection limit was 6.2 ng for anti-Stx1 and 12.5 ng for anti-Stx2, using an optical density of 0.1 as cut-off (Table 1). The equivalent cut-off was used for determining the reactivity and cross reactivity of MAbs and toxins. Anti-Stx1 (3E2) reacted with 0.1 μg of Stx1 up to 19.5 ng (Figure 2A), 2.5 μg of anti-Stx1 MAb was necessary to detect the same amount of Stx2 (Figure 2B). For anti-Stx2 (2E11) the reactivity with the Stx2 was up to 18.7 pg (Figure 2D) and 0.31 μg was necessary to detect Stx1 (Figure 2C). These results show that cross reactivity occurrs only in the presence of higher MAb concentrations, which are excessive to detected theirs respective toxins.

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