|SCOTCHER, MILES - Dupont Company
Submitted to: Journal of Immunological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/18/2012
Publication Date: 12/30/2012
Citation: He, X., Mcmahon, S.A., Skinner, C.B., Merrill, P.A., Scotcher, M., Stanker, L.H. 2013. Development and characterization of monoclonal antibodies against Shiga toxin 2 and their application for toxin detection in milk. Journal of Immunological Methods. 389(1-2):18-28. doi:10.1016/j.jim.2012.12.005.
Interpretive Summary: Although most E coli bacteria are beneficial to the health of the human gut, some strains of E coli produce toxins, including Shiga toxins (Stx). Shiga toxin producing E coli (STEC), including E coli O157:H7, are responsible for many outbreaks of foodborne illness. The deadly effects of Shiga toxin make it an especially important target for public health. While Stx comes in two types, Stx1 and Stx2, more disease is observed with STEC producing Stx2. In fact production of Stx2 is the most common and important factor associating an E coli strain with severe disease. Nonetheless, tests for detection of Stx are limited, and good tests for Stx2 in food are not commercially available. We have developed five new antibodies that specifically bind Stx2 in laboratory tests. Some of these antibodies also block the toxic effects of Stx2. In this paper we describe how we have used these new antibodies to make a test for Stx2 that is effective in milk.
Technical Abstract: Human infection of Shiga toxin producing Escherichia coli (STEC) is one of the most prevalent foodborne diseases. Shiga toxin 2 (Stx2) is the major contributor to hemolytic-uremic syndrome (HUS) and other systemic complications caused by STEC. Although outbreaks of HUS due to the consumption of dairy products occur frequently, very few reports are available on methods for the detection of Stx2 in milk. In this study, we describe the development of five high-affinity monoclonal antibodies (dissociation constants below nM range) against Stx2 using a genetic toxoid as an immunogen. These antibodies, designated ST1-1-3-6-2, ST1-18-2-4-1, ST1-103-3-3, ST1-122-3-3-2, and ST2-28-3-2-3 are IgG1 or IgG2a heavy-chain subclass with kappa light-chains and showed different specificities to variants of Stx2. Western blot analyses following SDS-PAGE demonstrate that mAbs ST1-18-2-4-1 and ST2-28-3-2-3 bind both the A- and B-subunits, while the other 3 mAbs bind the A-subunit of Stx2 only. All antibodies bound stronger to the native than to the denatured Stx2 except the mAb ST1-103-3-3, which bound equally well to both forms of the toxin. Of the five mAbs, ST2-28-3-2-3 was capable of neutralizing Stx2a mediated cytotoxicity in vero cells. A highly sensitive sandwich ELISA, capable of detecting as little as 10 pg/mL of Stx2a in milk, was developed using mAb pair ST1-1-3-6-2 and ST1-18-2-4-1. Such an assay is useful for early recognition of STEC contamination in food and prompt implementation of control measures to prevent outbreaks.