|KHALIL, ROWAIDA - Alexandria University Of Egypt|
|KONG, QUILIAN - Shanghai Academy Of Agricultural Sciences|
Submitted to: mSphere
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/3/2016
Publication Date: 2/17/2016
Publication URL: http://handle.nal.usda.gov/10113/63153
Citation: Skinner, C.B., Patfield, S.A., Khalil, R., Kong, Q., He, X. 2016. New monoclonal antibodies against a novel subtype of Shiga toxin 1 produced by Enterobacter cloacae and their use in analysis of human serum. mSphere. 1(1):e00099-15.
Interpretive Summary: Shiga toxins (Stx) are among the most clinically important virulence factors for Shigella and enterohemhorragic Escherichia coli (EHEC). There are many varieties of Stx, and they are diverse with respect to their sequence and pathogenicity. Although Stx1a and Stx2a are the most common and widely distributed types of Shiga toxin, new variants of Stx are continually emerging. These new variants of Stx can be challenging to detect, since most Stx detection kits are optimized for the detection of Stx1a and Stx2a. The recently discovered Stx1e, found in an atypical host (Enterobacter cloacae, in a clinical sample) is undetectable by many Stx assays. To formulate new assays for the detection of Stx1e, we generated four new monoclonal antibodies that recognize this Stx subtype. All four of these new antibodies recognize the A subunit of Stx1e. Using the best of these antibodies and an anti-Stx1 polyclonal antibody, we generated ELISA assays capable of detecting Stx1e at concentrations as low as 4.8 pg/mL. This assay is also compatible with a human serum matrix with a limit of detection of 53.6 pg/mL, suggesting that it may have utility for the clinical detection and diagnosis of Stx1e-associated infections.
Technical Abstract: Shiga toxin (Stx) is a major virulence factor for several bacterial pathogens that cause potentially fatal illness, including Escherichia coli and Shigella spp. The continual emergence of new subtypes of Stxs presents challenges in clinical diagnosis of infections caused by Shiga toxin-producing organisms. In this study, we report the development of four new monoclonal antibodies against Stx1e, a novel subtype of Stx1 which was produced by an Enterobacter cloacae strain and had limited reactivity with existing anti-Stx1 antibodies. Western blot analysis indicates that these mAbs were Stx1-specific, bound to the A-subunit, and had distinct preferences to subtypes of Stx1. Of the four mAbs, Stx1e-2 was capable of partially neutralizing cytotoxicities derived from the Stx1e in Vero cells. ELISAs assembled using these high affinity monoclonal antibodies detected Stx1e at concentrations as low as 4.8 pg/mL in PBS buffer, 53.6 pg/mL in spiked human serum samples, and were also capable of distinguishing Stx1e-producing strains in enriched cultures. These assays may therefore have clinical value in diagnosing Stx1e-producing bacterial infection. Additionally, characteristics of Stx1e, such as the origin of stx1e genes, conditions for toxin expression, receptor binding, and cytotoxicity were investigated using the new antibodies developed in this study. This information should be useful for further understanding the clinical significance and prevalence of Stx1e-harboring E. cloacae and other organisms.