An improved method for the sensitive detection of Shiga toxin 2 in human serum

Authors: He, Xiaohua; ARDISSINO, GIANLUIGI - Fondazione Irccs Ca’ Granda Ospedale Maggiore Policlinico; Patfield, Stephanie; Cheng, Luisa; Silva, Christopher - Chris; BRIGOTTI, MAURIZIO - University Of Bologna

Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/29/2018
Publication Date: 1/31/2018
Publication URL: https://handle.nal.usda.gov/10113/6472270
Citation: He, X., Ardissino, G., Patfield, S.A., Cheng, L.W., Silva, C.J., Brigotti, M. 2018. An improved method for the sensitive detection of Shiga toxin 2 in human serum. Toxins. 10(2):59. https://doi.org/10.3390/toxins10020059.
DOI: https://doi.org/10.3390/toxins10020059

Interpretive Summary: Shiga toxin 2 is the major virulence factor for diarrhorea-associated hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children and HUS survivors may suffer from lasting kidney damage. Currently, there are no FDA-approved therapies to treat this disease. Stx2 has to enter the blood system before it can be delivered to target organs. Early detection of Stx2 in human serum is essential for disease management. However, detection of Stx2 (not Stx1) has been mostly unsuccessful due to the presence of Stx2-binding components in blood. In this study, we report the development of a new immunoassay for Stx2 and the effect of guanidinium chloride on the detection. Using our improved method, the recovery rate for Stx2 in human serum increased 4-fold compared with standard method. The effectiveness of the method for detection of Stx2 in human serum was also validated using samples from STEC-infected patients. A specific physical interaction between Stx2 and the human serum amyloid P protein (HuSAP) in human serum samples was demonstrated by using coimmunoprecipitation and western blot analysis. This improved method will be useful to study the kinetic of Stx2 before and during the onset of HUS in STEC-infected patients.

Technical Abstract: Shiga toxins (Stx) produced by Stx-producing Escherichia coli (STEC) are virulence factors that is most closely associated with hemolytic uremic syndrome (HUS), a life-threatening complication of intestinal infections by STEC. Stx have to enter into the circulation system before they can be delivered to target organs and cause damage. The presence of Stx in sera could be a risk factor for HUS development. However, detection of Stx, particularly Stx2, has been difficult due to the presence of Stx2-binding components in human serum. Here, we report new ELISA-based methods for detection of Stx1 and Stx2 in human serum and the effect of guanidinium chloride on reducing ELISA inhibition with Stx2. The recovery rate for Stx2 was increased to 81% when serum samples were treated with guanidinium chloride at a concentration of 200 mM, compared with 17% without guanidinium chloride treatment. Co-immunoprecipitation results indicated a specific physical interaction between Stx2 and human serum amyloid P component (HuSAP) in human serum samples. However, our in vitro study demonstrated that the inhibition from HuSAP for detection of Stx2 was 35%, much less than 83% from human serum, suggesting there may exist other factors interacting with Stx2 in human serum besides HuSAP. This study indicates that treatment of serum samples with guanidinium chloride may be useful for early detection of Stx2 in human serum, thereby preventing severe complications associated with HUS even in the presence of various Stx2 binding components.