Interaction between Shiga Toxin and Monoclonal Antibodies: Binding Characteristics and in Vitro Neutralizing Abilities

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.

Diarrheagenic Escherichia coli

“…Sensitivities and specificities vary according to the test format and the manufacturer 199, 200, 201, 202, 203, 204, 205. Nevertheless, the standard by which each manufacturer evaluates its tests also varies; therefore, a direct comparison of performance characteristics of various immunoassays has not been performed 198, 206, 207. Moreover, these commercially available tests are not affordable for developing countries.…”

“…Moreover, these commercially available tests are not affordable for developing countries. Thus, to outline this, previous works have established different formats of immunoassays, employing either a mixture of rabbit anti-Stx1 and anti-Stx2 sera by indirect ELISA or polyclonal and monoclonal antibodies in a capture ELISA assay for the detection of STEC 207, 208, 209. The standardized methods are reproducible, fast, easy to perform, showing high sensitivity in detecting Stx by capture ELISA, even in low-producing isolates.…”

Recent Advances in Understanding Enteric Pathogenic Escherichia coli

“…Monoclonal antibodies (MAbs) have been shown to reduce the cytotoxicity of Stx1 and Stx2 in vitro (381), while Urtoxazumab, a humanized MAb against Stx2, was shown to be safe in a phase 1 study (382). Similarly, phase 1 clinical trials of chimeric monoclonal Stx1-and Stx2-neutralizing antibodies were also shown to be generally well tolerated in injected volunteers (383,384).…”

Anticorpo recombinante anti-intimina: uma ferramenta para o diagnóstico rápido de Escherichia coli enteropatogênica e de Escherichia coli enterohemorrágica.

“…2006) e para a toxina Stx de STEC (MENDES-LEDESMA et al, 2008;ROCHA et al, 2012 O sistema mais comumente utilizado para a expressão de proteínas heterólogas é o que utiliza E. coli como célula hospedeira (LEWIN, 1994(LEWIN, , 1997STRYER, 1988;ZAHA et al, 1996). Este sistema é amplamente difundido deviso à facilidade e baixo custo de se cultivar esta bactéria, e pela reprodutibilidade e abundância de proteínas que a mesma produz.…”