Staphylococcus aureus is an important bacterial pathogen that produces a range of Staphylococcal Enterotoxins (SEs) which cause gastroenteritis and superantigen activation of T cells, the mechanism of which is not well understood. The ability to rapidly detect and quantify SEs is very important in order to learn the causes of staphylococcal outbreaks and to stop similar outbreaks in the future. Enzyme-linked immunosorbent assays (ELISAs) have been developed for detection of several SEs. However, these immunological methods cannot distinguish between active and inactive toxin. It is known that interferon-gamma (IFN-γ) expressed in response to stimulation by SEs contributes significantly to the pathogenesis of S. aureus infection. Nonetheless, the cellular source of IFN-γ is still unclear and the contributions of the specific splenocyte types. In our effort to understand the immunologic response to Staphylococcal Enterotoxin A (SEA) exposure, we studied IFN-γ production in mouse splenocytes. We demonstrated that short term ex vivo exposure of splenocytes or primary naïve CD4⁺ T-cells to biologically active SEA induces differential expression of IFN-γ mRNA in a time and dose dependent manner and the expression levels reflect the levels of IFN-γ secreted protein. Positive isolated CD4⁺ T-cells accounted for only 10% of IFN-γ production. We also demonstrate that expression of IFN-γ can be used for rapid quantitative analysis of active SEA with a detection limit of 1 ng/mL.

In this study, sixteen unique staphylococcal enterotoxin B (SEB)-reactive nanobodies (nbs), including ten monovalent and six bivalent nbs, were developed. All characterized nbs were highly specific for SEB and did not cross-react with other staphylococcal enterotoxins (SE). Several formats of highly sensitive enzyme-linked immunosorbent assays (ELISAs) were established using SEB nbs and a polyclonal antibody (pAb). The lowest limit of detection (LOD) reached 50 pg/mL in PBS. When applied to an ELISA to detect SEB-spiked milk (a commonly contaminated foodstuff), a LOD as low as 190 pg/mL was obtained. The sensitivity of ELISA was found to increase concurrently with the valency of nbs used in the assay. In addition, a wide range of thermal tolerance was observed among the sixteen nbs, with a subset of nbs, SEB-5, SEB-9, and SEB-6², retaining activity even after exposure to 95 °C for 10 min, whereas the conventional monoclonal and polyclonal antibodies exhibited heat-labile properties. Several nbs demonstrated a long shelf-life, with one nb (SEB-9) retaining 93% of its activity after two weeks of storage at room temperature. In addition to their usage in toxin detection, eleven out of fifteen nbs were capable of neutralizing SEB’s super-antigenic activity, demonstrated by their inhibition on IL-2 expression in an ex vivo human PBMC assay. Compared to monoclonal and polyclonal antibodies, the nbs are relatively small, thermally stable, and easy to produce, making them useful in applications for sensitive, specific, and cost-effective detection and management of SEB contamination in food products.

Aflatoxin-producing fungi contaminate food and feed during pre-harvest, storage and processing periods. Once consumed, aflatoxins (AFs) accumulate in tissues, causing illnesses in animals and humans. Most human exposure to AF seems to be a result of consumption of contaminated plant and animal products. The policy of blending and dilution of grain containing higher levels of aflatoxins with uncontaminated grains for use in animal feed implicitly assumes that the deleterious effects of low levels of the toxins are linearly correlated to concentration. This assumption may not be justified, since it involves extrapolation of these nontoxic levels in feed, which are not of further concern. To develop a better understanding of the significance of low dose effects, in the present study, we developed quantitative methods for the detection of biologically active aflatoxin B₁ (AFB1) in Vero cells by two independent assays: the green fluorescent protein (GFP) assay, as a measure of protein synthesis by the cells, and the microculture tetrazolium (MTT) assay, as a measure of cell viability. The results demonstrate a non-linear dose-response relationship at the cellular level. AFB1 at low concentrations has an opposite biological effect to higher doses that inhibit protein synthesis. Additional studies showed that heat does not affect the stability of AFB1 in milk and that the Vero cell model can be used to determine the presence of bioactive AFB1 in spiked beef, lamb and turkey meat. The implication of the results for the cumulative effects of low amounts of AFB1 in numerous foods is discussed.

Abrin is a highly potent and naturally occurring toxin produced in the seeds of Abrus precatorius (Rosary Pea) and is of concern as a potential bioterrorism weapon. There are many rapid and specific assay methods to detect this toxic plant protein, but few are based on detection of toxin activity, critical to discern biologically active toxin that disables ribosomes and thereby inhibits protein synthesis, producing cytotoxic effects in multiple organ systems, from degraded or inactivated toxin which is not a threat. A simple and low-cost CCD detector system was evaluated with colorimetric and fluorometric cell-based assays for abrin activity; in the first instance measuring the abrin suppression of mitochondrial dehydrogenase in Vero cells by the MTT-formazan method and in the second instance measuring the abrin suppression of green fluorescent protein (GFP) expression in transduced Vero and HeLa cells. The limit of detection using the colorimetric assay was 10 pg/mL which was comparable to the fluorometric assay using HeLa cells. However, with GFP transduced Vero cells a hundred-fold improvement in sensitivity was achieved. Results were comparable to those using a more expensive commercial plate reader. Thermal inactivation of abrin was studied in PBS and in milk using the GFP-Vero cell assay. Inactivation at 100 °C for 5 min in both media was complete only at the lowest concentration studied (0.1 ng/mL) while treatment at 63 °C for 30 min was effective in PBS but not milk.

Staphylococcus aureus is a major bacterial pathogen which causes clinical infections and food poisoning. This bacterium produces a group of twenty-one enterotoxins (SEs). These enterotoxins have two separate but related biological activities. They cause gastroenteritis and function as superantigens that activate large numbers of T cells. The current method for detection of enterotoxins activity is an in vivo monkey or kitten bioassay; however, this method is not practical to test on a large number of samples. Several immunological assays have been developed however, but these assays cannot distinguish between active toxin which causes food poisoning and inactive toxin, which can bind antibody, but shows no toxicity. The current study demonstrates that short term ex vivo exposure of primary naïve CD4⁺ T-cells or splenocytes to SEA induces differential expression and secretion of tumor necrosis factor (TNF) protein. We used immunomagnetic beads coated with anti-SEA antibody to specifically isolate SEA from food. After the eluted toxin was added to the cells SEA biological activity was measured by quantifying TNF protein expression or secretion.