Streptococcal pyrogenic exotoxin B is a superantigen that induces murine splenocyte proliferation and secretion of IL-2 and IFN-γ ex vivo.

Streptococcus pyogenes is a significant human pathogen, producing a range of virulence factors including streptococcal pyrogenic exotoxin B (SpeB) that is associated with foodborne outbreaks. It was only known that this cysteine protease mediates cleavage of transmembrane proteins to permit bacterial penetration and is found in 25% of clinical isolates from Streptococcal Toxic Shock Syndrome (STSS) patients with extreme inflammation. Its interaction with host and streptococcal proteins has been well characterized but doubt remains about whether it constitutes a superantigen. In this study for the first time, it is shown that SpeB acts as a superantigen, similarly to other known superantigens like SEA or SPE-C, by inducing proliferation of murine splenocytes and cytokine secretion, primarily of interleukin-2 (IL-2) as shown by cytometric bead array analysis. IL-2 secretion was confirmed by enzyme-linked immunoassay (ELISA) as well as secretion of interferon-γ (IFN-γ). The ELISA assay showed a dose-dependent relationship between SpeB concentration in splenocyte cells and IL-2 secretion levels, and it was shown that SpeB retains activity in milk pasteurized for 30 minutes at 63°C.

A Sensitive, Cell-Based Assay for Measuring Low-Level Biological Activity of α-Amanitin.

α-Amanitin is one of the primary toxins produced by the poisonous mushroom genus, Amanita. Because it is odorless and tasteless, it is an important cause of death from the consumption of misidentified mushrooms. To study the thermal stability of α-amanitin, novel cell-based assays were developed to measure the toxin's activity, based on the inhibition of RNA polymerase II by α-amanitin. First, an MTT-formazan cell viability assay was used to measure the biological activity of α-amanitin through the inhibition of cellular activity. This method can detect 10 µg/mL of α-amanitin in a time-dependent manner. Second, a more sensitive quantitative PCR approach was developed to examine its inhibition of viral replication. The new RT-qPCR assay enabled the detection of 100 ng/mL. At this level, α-amanitin still significantly reduced adenovirus transcription. Third, a simpler GFP expression-based assay was developed with an equal sensitivity to the RT-qPCR assay. With this assay, aqueous α-amanitin heated at 90 °C for 16 h or treated in the microwave for 3 min retained its biological activity when tested in HEK293 cells, but a slight reduction was observed when tested in Vero cells. Beyond detecting the activity of α-amanitin, the new method has a potential application for detecting the activity of other toxins that are RNA polymerase inhibitors.

Development of Thermally Stable Nanobodies for Detection and Neutralization of Staphylococcal Enterotoxin B.

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.

T-cell receptor Vß8 for detection of biologically active streptococcal pyrogenic exotoxin type C.

Streptococcus pyogenes is an important human pathogen, commonly spread by airborne droplets but also by ingestion of contaminated food. Apart from causing infection, this pathogen produces 13 distinct types of streptococcal pyrogenic exotoxins (SPE). The current method for detection cannot distinguish between the biologically active form of SPE that has been reported to cause foodborne outbreaks and the inactivated toxin that poses no health risk. To measure the biological activity of SPE type C (SPE-C), one such toxin that was linked to foodborne outbreaks associated with milk and milk products, we developed a cell-based assay that can discern between biologically active and inactive SPE-C. To the best of our knowledge, this is the first showing that SPE-C activates T-cells expressing Vß8. With this finding, we used a T-cell line natively expressing Vß8 that was genetically engineered to also express the luciferase reporter gene under the regulation of nuclear factor of activated T-cells response element in combination with a B-cell line to present the recombinant SPE-C (rSPE-C) toxin via major histocompatibility complex (MHC) class II to the Vß8 T-cell receptor (TCR) in an assay to detect and to discern between biologically active and inactive rSPE-C. By using this system, we demonstrated that SPE-C induced significant IL-2 secretion after 72 h and visible light emission after only 5 h, doubling by 24 h. We utilize this finding to assess the specificity of the assay and the effect of pasteurization on SPE-C activity. We observed no cross-reactivity with SPE-B and significant loss of SPE-C biological activity in spiked phosphate-buffered saline while SPE-C spiked into milk is heat stable. Once SPE-C has formed, it is infeasible to eliminate it from milk by thermal treatment.

Effect of an Eco-Friendly Cuminaldehyde Guanylhydrazone Disinfectant on Shiga Toxin Production and Global Transcription of Escherichia coli.

Antimicrobials have been important medicines used to treat various infections. However, some antibiotics increase the expression of Shiga toxin (Stx). Also, the pervasive use of persistent antibiotics has led to ecotoxicity and antibiotic resistance. In this study, a newly developed broad-spectrum and reversible antibiotic (guanylhydrazone disinfectant) was evaluated for its antibiotic activity and effects on Stx production and global transcription of bacteria. No Stx induction was observed in 25 Shiga toxin-producing E. coli (STEC) isolates treated with a sublethal concentration of the guanylhydrazone. A differential gene expression study comparing two guanylhydrazone-treated to non-treated E. coli strains indicated that the expression of a group of stress-responsive genes were enhanced. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that guanylhydrazone treatment significantly downregulated the pathways of ribosome and flagellar assembly in both pathogenic and non-pathogenic strains and differentially regulated some pathways essential for bacteria to maintain cell shape and gain survival advantage in two strains. In addition, upregulation of antibiotic resistant genes related to the multidrug efflux system and virulence genes coding for colibactin, colicin, and adhesin was observed in strains treated with the disinfectant. The knowledge obtained in this study contributes to our understanding of the mode of this disinfectant action and facilitates our effort to better use disinfectants for STEC treatments.

Evaluation of a Witch Hazel Extract for the Potential Prebiotic and Protective Effect on Select Lactiplantibacillus plantarum (Prev. Lactobacillus plantarum) Strains.

Witch hazel extract has been evaluated in prior studies demonstrating the phenolic-mediated biofilm inhibition, toxin production inhibition, and growth inhibition in Staphylococcus aureus. In this study, we are evaluating the possible prebiotic and protective effect of witch hazel extract on select probiotic Lactiplantibacillus plantarum strains, namely L. plantarum LP 10241 and L. plantarum LPBAA-793. When the prebiotic effect was evaluated, we observed that the tested extract had prebiotic effect at the higher tested dose (0.5%) on LPBAA-793 strain (8.7 log CFU/mL after 18 h compared to 5.1 log CFU/mL with the control) and on LP 10241 strain (7.7 log CFU/mL after 18 h compared to 4.4 log CFU/mL with the control). For the evaluation of the protective effect of witch hazel extract on the select strains, we subjected nutrient depletion stress under aerobic conditions and monitored the cell death with and without addition of witch hazel extract. We observed that the tested extract had a significant protective effect on LPBAA-793 strain (4 log CFU/mL after 12 days, compared to no growth with control) and a slighter protective effect against LP 10241 strains (6.3 log CFU/mL in day 2 compared to 4.3 log CFU/mL with control). The results from this research provide for the first time the rationale that while witch hazel extract has significant antimicrobial, anti-toxin production and anti-biofilm activities on pathogenic microorganisms, it might play an important and positive role on health-beneficial probiotic bacteria.

The Effect of Tannin-Rich Witch Hazel on Growth of Probiotic Lactobacillus plantarum.

Probiotic bacteria help maintain microbiome homeostasis and promote gut health. Maintaining the competitive advantage of the probiotics over pathogenic bacteria is a challenge, as they are part of the gut microbiome that is continuously exposed to digestive and nutritional changes and various stressors. Witch hazel that is rich in hamamelitannin (WH, whISOBAXTM) is an inhibitor of growth and virulence of pathogenic bacteria. To test for its effect on probiotic bacteria, WH was tested on the growth and biofilm formation of a commercially available probiotic Lactobacillus plantarum PS128. As these bacteria are aerotolerant, the experiments were carried out aerobically and in nutritionally inadequate/poor (nutrient broth) or adequate/rich (MRS broth) conditions. Interestingly, despite its negative effect on the growth and biofilm formation of pathogenic bacteria such as Staphylococcus epidermidis, WH promotes the growth of the probiotic bacteria in a nutritionally inadequate environment while maintaining their growth under a nutritionally rich environment. In the absence of WH, no significant biofilm is formed on the surfaces tested (polystyrene and alginate), but in the presence of WH, biofilm formation was significantly enhanced. These results indicate that WH may thus be used to enhance the growth and survival of probiotics.

Ex Vivo and In Vitro Methods for Detection of Bioactive Staphylococcal Enterotoxins.

Staphylococcus aureus is a major bacterial cause of clinical infections and foodborne illnesses.Through the synthesis of a group of Staphylococcal enterotoxins (SEs), gastroenteritis occurs and the SEs function as superantigens to massively activate T cells. The ability to rapidly detect and quantify SEs is imperative in order to learn the causes of staphylococcal outbreaks and to stop similar outbreaks in the future. Also, the ability to discern active toxin is essential for development of food treatment and processing methods. Here, we discuss the various methodologies for detection and analysis of SEs.

Prevalence and Genetic Analysis of Chromosomal mcr-3/7 in Aeromonas From U.S. Animal-Derived Samples.

The prevalence of mcr-positive bacteria in 5,169 domestic animal-derived samples collected by USDA Food Safety and Inspection Service between October 2018 and May 2019 was investigated. A procedure including enriched broth culture and real-time PCR targeting mcr-1 to mcr-8 were used for the screening. Fifteen positive isolates were identified, including one plasmid-borne mcr-1-positive Escherichia coli strain, EC2492 (reported elsewhere) and 14 mcr-3/7-positive strains from poultry (1), catfish (2), and chicken rinse (11) samples, resulting in an overall prevalence of mcr-positive bacteria 0.29% in all meat samples tested. Analysis of 16S rRNA and whole genome sequences revealed that all 14 strains belonged to Aeromonas. Data from phylogenetic analysis of seven housekeeping genes, including gyrB, rpoD, gyrA, recA, dnaJ, dnaX, and atpD, indicated that nine strains belonged to Aeromonas hydrophila and five strains belonged to Aeromonas jandaei. Antimicrobial tests showed that almost all mcr-positive strains exhibited high resistance to colistin with MICs ≥ 128mg/L, except for one A. jandaei strain, which showed a borderline resistance with a MIC of 2 mg/L. A segment containing two adjacent mcr-3 and mcr-3-like genes was found in two A. hydrophila and one A. jandaei strains and a variety of IS-like elements were found in the flanking regions of this segment. A mcr-3-related lipid A phosphoethanolamine transferase gene was present in all 14 Aeromonas strains, while an additional mcr-7-related lipid A phosphoethanolamine transferase gene was found in 5 A. jandaei strains only. In addition to mcr genes, other antimicrobial resistance genes, including bla OXA-12/OXA-724, aqu-2, tru-1, cepS, cphA, imiH, ceph-A3, ant(3″)-IIa, aac(3)-Via, and sul1 were observed in chromosomes of some Aeromonas strains. The relative high prevalence of chromosome-borne mcr-3/7 genes and the close proximity of various IS elements to these genes highlights the need for continued vigilance to reduce the mobility of these colistin-resistance genes among food animals.

Human Leukemia T-Cell Lines as Alternatives to Animal Use for Detecting Biologically Active Staphylococcal Enterotoxin Type B.

Staphylococcal enterotoxin type B (SEB) is associated with food poisoning. Current methods for the detection of biologically active SEB rely upon its ability to cause emesis when administered to live kittens or monkeys. This technique suffers from poor reproducibility and low sensitivity and is ethically disfavored over concerns for the welfare of laboratory animals. The data presented here show the first successful implementation of an alternative method to live animal testing that utilizes SEB super-antigenic activity to induce cytokine production for specific novel cell-based assays for quantifiable detection of active SEB. Rather than using or sacrificing live animals, we found that SEB can bind to the major histocompatibility complex (MHC) class II molecules on Raji B-cells. We presented this SEB-MHC class II complex to specific Vß5.3 regions of the human T-cell line HPB-ALL, which led to a dose-dependent secretion of IL-2 that is capable of being quantified and can further detect 10 pg/mL of SEB. This new assay is 100,000 times more sensitive than the ex vivo murine splenocyte method that achieved a detection limit of 1 µg/mL. The data presented here also demonstrate that SEB induced proliferation in a dose-dependent manner for cells obtained by three different selection methods: by splenocyte cells containing 22% of CD4+ T-cells, by CD4+ T-cells enriched to >90% purity by negative selection methods, and by CD4+ T-cells enriched to >95% purity by positive selection methods. The highly enriched and positively isolated CD4+ T-cells with the lowest concentration of antigen-presenting cells (APC) (below 5%) provided higher cell proliferation than the splenocyte cells containing the highest concentration of APC cells.

Validation of a Cell-Based Assay for Detection of Active Shiga Toxins Produced by Escherichia coli in Water.

Shiga toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases, including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Almost 5% of STEC infections result from waterborne exposures, yet there is no test listed in the EPA's current Selected Analytical Methods for the detection of active Shiga toxins (Stxs) in water. In this study, a HeLa cell-based assay is validated for the detection of metabolically active Stxs produced by STEC in water, including tap, bottled, and pond water. Active Stxs are detected even when the number of Stx-producing bacteria is less than 0.4 CFU/mL and the assay performance is not affected by background flora or chlorine in the water. This assay is not only as simple and affordable as cell-free assays but also detects active holotoxins without the use of live animals. In addition, the assay is designed for use in multi-well formats, making it ideal for high-throughput screening of water samples and therefore useful for environmental public health surveillance programs to reduce human risk of infection with STEC.

Quantitative bioluminescence assay for measuring Bacillus cereus nonhemolytic enterotoxin complex.

Bacillus cereus is a foodborne pathogen causing emesis and diarrhea in those affected. It is assumed that the non-hemolytic enterotoxin (Nhe) plays a key role in B. cereus induced diarrhea. The ability to trace Nhe activity is important for food safety. While assays such as PCR and ELISA exist to detect Nhe, those methods cannot differentiate between active and inactive forms of Nhe. The existing rabbit ileal loop bioassay used to detect Nhe activity is ethically disfavored because it uses live experimental animals. Here we present a custom built low-cost CCD based luminometer and applied it in conjunction with a cell-based assay using Vero cells transduced to express the luciferase enzyme. The activity of Nhe was measured as its ability to inhibit synthesis of luciferase as quantified by reduction of light emission by the luciferase reaction. Emitted light intensity was observed to be inversely proportional to Nhe concentration over a range of 7 ng/ml to 125 ng/ml, with a limit of detection of 7 ng/ml Nhe.

whISOBAXTM Inhibits Bacterial Pathogenesis and Enhances the Effect of Antibiotics.

As bacteria are becoming more resistant to commonly used antibiotics, alternative therapies are being sought. whISOBAX (WH) is a witch hazel extract that is highly stable (tested up to 2 months in 37 °C) and contains a high phenolic content, where 75% of it is hamamelitannin and traces of gallic acid. Phenolic compounds like gallic acid are known to inhibit bacterial growth, while hamamelitannin is known to inhibit staphylococcal pathogenesis (biofilm formation and toxin production). WH was tested in vitro for its antibacterial activity against clinically relevant Gram-positive and Gram-negative bacteria, and its synergy with antibiotics determined using checkerboard assays followed by isobologram analysis. WH was also tested for its ability to suppress staphylococcal pathogenesis, which is the cause of a myriad of resistant infections. Here we show that WH inhibits the growth of all bacteria tested, with variable efficacy levels. The most WH-sensitive bacteria tested were Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecium and Enterococcus faecalis, followed by Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Streptococcus agalactiae and Streptococcus pneumoniae. Furthermore, WH was shown on S. aureus to be synergistic to linezolid and chloramphenicol and cumulative to vancomycin and amikacin. The effect of WH was tested on staphylococcal pathogenesis and shown here to inhibit biofilm formation (tested on S. epidermidis) and toxin production (tested on S. aureus Enterotoxin A (SEA)). Toxin inhibition was also evident in the presence of subinhibitory concentrations of ciprofloxacin that induces pathogenesis. Put together, our study indicates that WH is very effective in inhibiting the growth of multiple types of bacteria, is synergistic to antibiotics, and is also effective against staphylococcal pathogenesis, often the cause of persistent infections. Our study thus suggests the benefits of using WH to combat various types of bacterial infections, especially those that involve resistant persistent bacterial pathogens.

CCD Based Detector for Detection of Abrin Toxin Activity.

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.

Witch Hazel Significantly Improves the Efficacy of Commercially Available Teat Dips.

Bovine intramammary infections (IMIs) are the main cause of economic loss in milk production. Antibiotics are often ineffective in treating infections due to antimicrobial resistance and the formation of bacterial biofilms that enhance bacterial survival and persistence. Teat dips containing germicides are recommended to prevent new IMIs and improve udder health and milk quality. IMIs are often caused by staphylococci, which are Gram-positive bacteria that become pathogenic by forming biofilms and producing toxins. As a model for a teat dip (DIP), the BacStop iodine-based teat dip (DIP) was used. Witch hazel extract (whISOBAX (WH)) was tested because it contains a high concentration of the anti-biofilm/anti-toxin phenolic compound hamamelitannin. We found that the minimal inhibitory or bactericidal concentrations of DIP against planktonic S. epidermidis cells increased up to 160fold in the presence of WH, and that DIP was 10-fold less effective against biofilm cells. While both DIP and WH are effective in inhibiting the growth of S. aureus, only WH inhibits toxin production (tested for enterotoxin-A). Importantly, WH also significantly enhances the antibacterial effect of DIP against Gram-negative bacteria that can cause IMIs, like Escherichia coli and Pseudomonas aeruginosa. Put together, these results suggest that the antibacterial activity of DIP combined with WH is significantly higher, and thus have potential in eradicating bacterial infections, both in acute (planktonic-associated) and in chronic (biofilm-associated) conditions.

In-Vitro Inhibition of Staphylococcal Pathogenesis by Witch-Hazel and Green Tea Extracts.

whISOBAX (WH), an extract of the witch-hazel plant that is native to the Northeast coast of the United States, contains significant amounts of a phenolic compound, Hamamelitannin (HAMA). Green tea (GT) is a widely consumed plant that contains various catechins. Both plants have been associated with antimicrobial effects. In this study we test the effects of these two plant extracts on the pathogenesis of staphylococci, and evaluate their effects on bacterial growth, biofilm formation, and toxin production. Our observations show that both extracts have antimicrobial effects against both strains of S. aureus and S. epidermidis tested, and that this inhibitory effect is synergistic. Also, we confirmed that this inhibitory effect does not depend on HAMA, but rather on other phenolic compounds present in WH and GT. In terms of biofilm inhibition, only WH exhibited an effect and the observed anti-biofilm effect was HAMA-depended. Finally, among the tested extracts, only WH exhibited an effect against Staphylococcal Enterotoxin A (SEA) production and this effect correlated to the HAMA present in WH. Our results suggest that GT and WH in combination can enhance the antimicrobial effects against staphylococci. However, only WH can control biofilm development and SEA production, due to the presence of HAMA. This study provides the initial rationale for the development of natural antimicrobials, to protect from staphylococcal colonization, infection, or contamination.

T cell Receptor Vß9 in Method for Rapidly Quantifying Active Staphylococcal Enterotoxin Type-A without Live Animals.

Staphylococcal food poisoning is a result of ingestion of Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus. Staphylococcal enterotoxin type A (SEA) is the predominant toxin produced by S. aureus strains isolated from food-poisoning outbreak cases. For public safety, assays to detect and quantify SEA ideally respond only to the active form of the toxin and this usually means employing disfavored live animal testing which suffers also from poor reproducibility and sensitivity. We developed a cell-based assay for SEA quantification in which biologically-active SEA is presented by Raji B-cells to CCRF-CEM T-cells resulting in internalization of Vß9 within 2 hours with dose dependency over a 6-log range of SEA concentrations. This bioassay can discern biologically active SEA from heat-inactivated SEA and is specific to SEA with no cross reactivity to the homologically-similar SED or SEE. In this study, we terminated any ongoing biochemical reactions in accessory cells while retaining the morphology of the antigenic sites by using paraformaldehyde fixation and challenged the current model for mechanism of action of the SEA superantigen. We demonstrated for the first time that although fixed, dead accessory cells, having no metabolic functions to process the SEA superantigen into short peptide fragments for display on their cell surface, can instead present intact SEA to induce T-cell activation which leads to cytokine production. However, the level of cytokine secretion induced by intact SEA was statistically significantly lower than with viable accessory cells, which have the ability to internalize and process the SEA superantigen.

Synthesis of pyrrolidine-based hamamelitannin analogues as quorum sensing inhibitors in Staphylococcus aureus.

Interfering with bacterial cell-to-cell communication is a promising strategy to combat antimicrobial resistance. The natural product hamamelitannin and several of its analogues have been identified as quorum sensing inhibitors. In this paper the synthesis of pyrrolidine-based analogues of a more lead-like hamamelitannin analogue is reported. A convergent synthetic route based on a key ring-closing metathesis reaction was developed and delivered the pyrrolidine analogue in 17 steps in high yield. Chemoselective derivatization of the pyrrolidine nitrogen atom resulted in 6 more compounds. The synthesized compounds were evaluated in a biofilm model, but were all inactive.

Alternative to Animal Use for Detecting Biologically Active Staphylococcal Enterotoxin Type A.

Staphylococcal enterotoxins (SEs) are a food safety concern. Existing methods for biologically active SE detection rely on the emetic response in live kittens or monkeys. This method suffers from low sensitivity, poor reproducibility, and causes ethical concerns regarding the use of experimental animals. The Lautenberg Chemical Safety Act encourages the development and adoption of alternatives to testing on animals for chemical toxicity methodologies. In this study, we utilized the superantigenic effect of SE type A (SEA) and used an ex vivo bioassay as an alternative to live animal testing. We found that interleukin-2 (IL-2) secreted by splenocyte can be utilized for quantifiable detection of SEA in food products. To avoid food matrix interference and attenuation of signal, we separated SEA from spiked food products by employing immunomagnetic beads that were coated with an anti-SEA antibody. This ex vivo method has achieved the detection of 1 ng mL-1 of SEA, which is 107 times more sensitive than the existing live animal testing methods. However, this ex vivo bioassay requires sacrificing of mice. To overcome this limitation, we established a cell based in vitro assay using CCRF-CEM, a human CD4⁺ T-cell line, for the quantitative detection of SEA. Incubation of SEA with CCRF-CEM human T-cells and Raji cells led to quantifiable and dose dependent secretion of IL-2. This novel cell-based assay is highly specific to biologically active SEA, compared with the related SE toxin subtypes B, D, and E or heat inactivated SEA, which produce no secretion of IL-2. This is the first demonstration of an alternative assay that completely eliminates the use of animals for quantitative detection of active SEA.

Detection of Abrin Holotoxin Using Novel Monoclonal Antibodies.

Abrin, a member of the ribosome-inactivating protein family, is produced by the Abrus precatorius plant. Having the potential to pose a severe threat to both human and animal health, abrin is classified as a Select Agent by the U.S. Department of Health and Human Services. However, an immunoassay that is specific for intact abrin holotoxin has not yet been reported. In this study, seven new monoclonal antibodies (mAbs), designated as Abrin-1 through Abrin-7 have been developed. Isotyping analyses indicate these mAbs have IgG1, IgG2a, or IgG2b heavy-chains and kappa light-chains. Western blot analyses identified two abrin A-chain specific mAbs, Abrin-1 and Abrin-2, and four B-chain specific mAbs (Abrin-3, -5, -6, and -7). A sandwich enzyme-linked immunosorbent assay (ELISA), capable of detecting a mixture of abrin isoforms and agglutinins was developed using B-chain specific Abrin-3 for capture and A-chain specific Abrin-2 as detector. The ELISA is highly sensitive and detects 1 ng/mL of the abrin holotoxin in phosphate-buffered saline, nonfat milk, and whole milk, significantly below concentrations that would pose a health concern for consumers. This ELISA also detects native abrin in plant extracts with a very low background signal. The new abrin mAbs and ELISA should be useful for detecting this potent toxin in the milk supply chain and other complex matrices.