An Improved Method for the Sensitive Detection of Shiga Toxin 2 in Human Serum
Western Regional Research Center, U.S. Department of Agriculture, Agricultural Research Service, 800 Buchanan Street, Albany, CA 94710, USA
Center for HUS Control, Prevention and Management, Fondazione IRCCS Ca’Granda Ospedale Maggiore Policlinico, Via Commenda 9, 20122 Milano, Italy
Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Via San Giacomo 14, 40126 Bologna, Italy
Authors to whom correspondence should be addressed.
Toxins 2018, 10(2), 59; https://doi.org/10.3390/toxins10020059
Received: 18 November 2017 / Revised: 12 January 2018 / Accepted: 29 January 2018 / Published: 31 January 2018
(This article belongs to the Collection Rapid Detection of Bacterial Toxins)
Shiga toxins (Stx) released by Stx-producing E. coli (STEC) are virulence factors that are most closely associated with hemolytic uremic syndrome (HUS), a life-threatening complication of intestinal infections by STEC. Stx have to enter into the circulatory system before they are delivered to target organs and cause damage. The presence of Stx in sera could be a risk indicator for HUS development. However, the 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 the detection of Stx1 and Stx2 in human serum and the effect of guanidinium chloride on enhancing the sensitivity for the detection of Stx2. The recovery rate for Stx2 was 62% when Stx2-spiked serum samples were treated with guanidinium chloride at a concentration of 200 mM, in contrast to 17% without guanidinium chloride treatment. The effectiveness of guanidinium chloride treatment for the detection of Stx2 in human serum was validated using sera from STEC-infected patients. Coimmunoprecipitation results indicated a specific physical interaction between Stx2 and the human serum amyloid P component (HuSAP) in human serum samples. Our in vitro study demonstrated that the inhibition from HuSAP alone for the detection of Stx2 was only 20%, much less than 69.6% from human serum at Stx2 level 10 ng/mL, suggesting that there may be other factors that bind Stx2 in human serum. This study indicates that treatment of serum samples with guanidinium chloride may be useful for the early and sensitive detection of Stx2 in sera of STEC-infected patients, so preventive measures can be adopted in a timely manner.