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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

Nitrocellulose membranes containing purified toxins Shiga toxins (Stx)1 and Stx2. Immunoblotting reaction was carried out using anti-Stx1 MAb (A) and anti-Stx2 MAb (B). Apparent molecular weights found showed that the MAbs recognized their corresponding toxin. Arrows indicate toxin subunits.
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toxins-04-00729-f001: Nitrocellulose membranes containing purified toxins Shiga toxins (Stx)1 and Stx2. Immunoblotting reaction was carried out using anti-Stx1 MAb (A) and anti-Stx2 MAb (B). Apparent molecular weights found showed that the MAbs recognized their corresponding toxin. Arrows indicate toxin subunits.

Mentions: Secretory hybridomas of antibodies against Stx1 and Stx2 were obtained and subcloned by limiting dilution. Anti-Stx1 and anti-Stx2 MAbs produced by the selected clones (3E2 and 2E11, respectively), were classified as IgG1 and showed reactivity with their respective toxins by immunoblotting: anti-Stx1 MAb bound to the B subunit (Figure 1A) with a dissociation constant of 2.5 × 10−10 M (Table 1), while anti-Stx2 MAb bound to the A subunit (Figure 1B) with a dissociation constant of 6.1 × 10−10 M (Table 1).


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)

Nitrocellulose membranes containing purified toxins Shiga toxins (Stx)1 and Stx2. Immunoblotting reaction was carried out using anti-Stx1 MAb (A) and anti-Stx2 MAb (B). Apparent molecular weights found showed that the MAbs recognized their corresponding toxin. Arrows indicate toxin subunits.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

toxins-04-00729-f001: Nitrocellulose membranes containing purified toxins Shiga toxins (Stx)1 and Stx2. Immunoblotting reaction was carried out using anti-Stx1 MAb (A) and anti-Stx2 MAb (B). Apparent molecular weights found showed that the MAbs recognized their corresponding toxin. Arrows indicate toxin subunits.
Mentions: Secretory hybridomas of antibodies against Stx1 and Stx2 were obtained and subcloned by limiting dilution. Anti-Stx1 and anti-Stx2 MAbs produced by the selected clones (3E2 and 2E11, respectively), were classified as IgG1 and showed reactivity with their respective toxins by immunoblotting: anti-Stx1 MAb bound to the B subunit (Figure 1A) with a dissociation constant of 2.5 × 10−10 M (Table 1), while anti-Stx2 MAb bound to the A subunit (Figure 1B) with a dissociation constant of 6.1 × 10−10 M (Table 1).

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