Submitted to: Journal of Immunological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/19/2017
Publication Date: 4/20/2017
Publication URL: https://handle.nal.usda.gov/10113/5675927
Citation: He, X., Patfield, S.A., Rasooly, R., Mavrici, D. 2017. Novel monoclonal antibodies against Stx1d and 1e and their use for improvement of immunoassays. Journal of Immunological Methods. 44:52-56.
Interpretive Summary: Shiga toxin (Stx)-producing Escherichia coli (STEC) are noted for their association with food and water-linked outbreaks of Stx-mediated diseases. A possible complication from an infection is the hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children, and there is an 8% fatality rate for those individuals. Currently, there are no FDA-approved therapies in the United States to treat or prevent illness caused by STEC. Early detection of Stx is a crucial measure for disease management. In this study, we developed new monoclonal antibodies (mAbs) against two forms of Stx, Stx1d and Stx1e, and incorporated them into the existing commercial assay, Abraxis Stx1. It was demonstrated the new mAbs significantly improved the performance of the assay. The sensitivity of the test for Stx1d-producing STEC, was increased 3-fold, and for Stx1e-producing STEC was changed from undetectable to easily detectable. In addition, precision analysis indicated the improvement of the Abraxis Stx1 by our new mAbs is highly reproducible. This study demonstrates the new mAbs are valuable tools for improvement of commercial tests for detection of broader STEC strains.
Technical Abstract: Shiga toxins (Stxs) are major causative agents for bloody diarrhea and hemolytic uremic syndrome, a life-threatening disease in humans. No effective treatment is available. Early detection of Stxs in clinical samples is critical for disease management. As bacteria evolve, new Stxs are produced; therefore, methods used to identify them need to be improved as well. In this study, new monoclonal antibodies (mAbs) against Stx1d and 1e were developed and used to improve a commercial Stx1 test. Incorporation of the new mAbs into the Abraxis Stx1 test not only increased the assay sensitivity to Stx1d, but this toxin was conferred the ability to detect Stx1e, a newly identified subtype of Stx1 produced by an atypical Stx-producing bacterial strain, Enterobacter cloacae M12X01451, isolated from a clinical specimen. This toxin was not detectable using existing commercial kits. The signal to noise ratio (s/n) of the new assay was increased 3 fold for Stx1d, and 44 fold for Stx1e at toxin concentration of 10 ng/mL. The limit of detection (LOD) was 10 pg/mL for Stx1a, and 100 pg/mL for Stx1c, 1d and 1e. When used for bacterial strains, the sensitivity of the new assay was improved 2.5 to 60 fold depending on subtypes produced. In summary, high affinity mAbs against Stx1d and 1e were developed and incorporation of these mAbs into the Abraxis Stx1 test significantly enhanced the assay sensitivity and broadened the subtype-specificity. This improvement should be useful for reducing product recalls and disease mistreatment due to failures of detecting less common, but clinically important, subtypes of Stxs.