Development and characterization of monoclonal antibodies against Shiga toxin 2 and their application for toxin detection in milk

Human infection by Shiga toxin producing Escherichia coli (STEC) is one of the most prevalent foodborne diseases. Shiga toxin type 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 assays 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 recombinant toxoid as an immunogen. These antibodies, designated Stx2-1, Stx2-2, Stx2-3, Stx2-4, and Stx2-5 are IgG1 or IgG2a heavy-chain subclass with kappa light-chains, did not cross-react with Stx1 and showed different preferences to variants of Stx2. Western blot analyses demonstrate that mAbs Stx2-2 and Stx2-5 bind both the A- and B-subunits, whereas the other 3 mAbs bind the A-subunit of Stx2a only. All antibodies bound stronger to the native than to the denatured Stx2a except the mAb Stx2-3, which bound equally well to both forms of the toxin. Of the five mAbs, Stx2-5 was capable of neutralizing Stx2a mediated cytotoxicity in Vero cells. Highly sensitive ELISA and immuno-PCR assays, capable of detecting 1 and 0.01 pg/mL of Stx2a in milk, were developed using mAb pair Stx2-1 and Stx2-2. Such assays are useful for routine diagnosis of Stx2 contamination in milk production process, thus reducing the risk of STEC outbreaks.

► Five high-affinity monoclonal antibodies (mAbs) to the native Stx2 were developed.
► These antibodies showed different preferences to different variants of Stx2.
► Three mAbs bound the Stx2 A-subunit, two mAbs bound both the A- and B-subunits.
► One of the mAbs was capable of neutralizing the toxicity of Stx2a in Vero cells.
► Highly sensitive immunoassays were established to detect 10 fg/mL Stx2 in milk.