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Technologies for Detecting and Determining the Bioavailability of Bacterial Toxins


Provide toxicological data and analytical methodology for microbial toxins that will help ensure a safe food supply.

(1) Develop new assays for bacterial toxins and their variants, using immunological and other methods, with emphasis on applicability to practical problems facing the food industry and regulatory agencies. Develop new monoclonal antibody (mAb)-based assays for botulinum neurotoxins (BoNTs), non-toxic neurotoxin-associated proteins, and Shiga toxins (Stx), and optimize antibodies for biosensor applications. Develop methodology for detection of Shiga toxin-producing E. coli (STEC) and a multiplex bead-array assay for detecting Stx and STEC pathogenicity/virulence factors. Develop improved activity assay for staphylococcal enterotoxins.

(2) Calibrate in vitro methodology against established animal bioassays, and develop new data on the bioavailability of toxins, the impact of food processing on toxin activities, and the significance of antibody-mediated clearance on toxicity, especially via the oral route of intoxication. Determine the bioavailability of different botulinum neurotoxin serotypes. Validate new toxin assays using activity assays.

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Approach: For the first objective, the general approaches are to exploit immunoassays, especially enzyme-linked immunosorbent assay (ELISA), immuno-polymerase chain reaction (iPCR), and bead array assays because of their versatility, robustness, and sensitivity; and to develop activity assays. The mAbs developed for immunoassay will also have important utility for sample preparation and potential for diagnostic/therapeutic applications. Development of new toxin-specific mAbs will exploit a variety of immunogens, including toxoids and recombinant polypeptide chains corresponding to different domains of the toxin chains. Methodologies to optimize antibodies include the use of flow cytometry to test and select hybridoma cell lines. Structurally different antibodies such as IgY and single-chain antibodies will also be developed and compared to mAbs. Optimal capture/detection antibody pairs will be identified using ELISA and assay performance will be investigated with respect to robustness. Selected capture antibodies will be coupled to immunomagnetic beads for use in sample preparation. Assays will be evaluated in the food matrices of principal interest: milk, juices, liquid eggs, and ground meat and poultry products. For samples that produce a high background signal, matrix interference, or poor recovery, simple preparative methods will be tested, such as differential centrifugation, filtration, or immunomagnetic bead separations. Similar methodology will be used to develop antibodies and assays for accessory proteins found in toxin preparations as secreted by bacteria. Activity assays report active toxin. They will be especially useful to measure toxins in the presence of thermally inactivated and degraded proteins that are found in processed food samples. Assays that measure the activity of selected toxins (such as Stx and staphyloccocal enterotoxins) will utilize existing and new cell lines that are sensitive to active toxin. Suitable readout systems include cell lines that produce reporter molecules in response to toxin. For the second objective, the general approach is to relate variations in toxin structure to toxicity, bioavailability, and responses in detection systems. Bioassays and cell-based assays will be used to assess the impact of food processing on toxin activity and bioavailability. Dose-response and bioavailability will be determined for BoNT holotoxins and toxin complexes. We will determine the effect of accessory proteins on toxicity. The transit time for passage through the intestinal epithelium will be determined for holotoxin and toxin complexes in a model system, polarized colonic epithelial translocation assay. The protective effect of newly developed antibodies will be determined for various BoNTs. Assay validation is based on side-by-side comparison of samples in different assay systems. Active toxin concentration will be estimated by biochemical and cellular assays. Bioassays will be used for assessment of toxicity of unknown concentrations of toxins for comparison with in vitro assays, especially for toxin in raw and processed food matrices.
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Brandon, David
Carter, John Mark
Cheng, Luisa Wai Wai
He, Xiaohua
Hernlem, Bradley
Rasooly, Reuven
Stanker, Larry
Project Reports:
Published Journal Articles USDA ARS (NP 108):
Effects of electron-beam irradiation on blueberries inoculated with Escherichia coli and their nutritional quality and shelf life
Kong Q, Wu A, Qi W, Qi R, Carter JM, Rasooly R, He X.
Postharvest Biol Tec. 2014 Sep;95:28-35.
New High-Affinity Monoclonal Antibodies against Shiga Toxin 1 Facilitate the Detection of Hybrid Stx1/Stx2 In Vivo
Skinner C, Patfield S, Stanker LH, Fratamico P, He X.
PLoS One. 2014 Jun 10;9(6):e99854.
Quantitative analysis of staphylococcus enterotoxin A by differential expression of IFN-γ in splenocyte and CD4⁺ T-cells
Rasooly R, Hernlem BJ.
Sensors (Basel). 2014 May 20;14(5):8869-76.
Antibody interactions with Ricinus communis agglutinins studied by biolayer interferometry
Brandon DL, Adams L, Yang LL, Korn AM .
Anal Lett. 2014 Mar 5. [Epub ahead of print]
Atoxic Derivative of Botulinum Neurotoxin A as a Prototype Molecular Vehicle for Targeted Delivery to the Neuronal Cytoplasm
Vazquez-Cintron EJ, Vakulenko M, Band PA, Stanker LH, Johnson EA, Ichtchenko K.
PLoS One. 2014 Jan 22;9(1):e85517.
Thousand-fold fluorescent signal amplification for mHealth diagnostics
Balsam J, Rasooly R, Bruck HA, Rasooly A.
Biosens Bioelectron. 2014 Jan 15;51:1-7.
Prediction of B-cell epitopes in Listeriolysin O, a cholesterol-dependent cytolysin secreted by Listeria monocytogenes
Jones MS, Carter JM .
Adv Bioinformatics. 2014 Jan 2;871676:.
Immunosorbent analysis of ricin contamination in milk using colorimetric, chemiluminescent and electrochemiluminescent detection
Brandon DL, Korn AM, Yang LL.
Food Agric Immunol. 2014;25(2):160-72.
Microwave heating inactivates shiga toxin (Stx2) in reconstituted fat-free milk and adversely affects the nutritional value of cell culture medium
Rasooly R, Hernlem B, He X, Friedman M.
J Agric Food Chem. 2014;62(14):3301-5.
Prediction of B-cell epitopes in listeriolysin O, a cholesterol dependent cytolysin secreted by Listeria monocytogenes
Jones MS, Carter JM.
Adv Bioinformatics. 2014;2014:.
Comprehensive laboratory evaluation of a highly specific lateral flow assay for the presumptive identification of ricin in suspicious white powders and environmental samples
Hodge DR, Prentice KW, Ramage JG, Prezioso S, Gauthier C, Swanson T, Hastings R, Basavanna U, Datta S, Sharma SK, Garber EA, Staab A, Pettit D, Drumgoole R, Swaney E, Estacio PL, Elder IA, Kovacs G, Morse BS, Kellogg RB, Stanker L, Morse SA, Pillai SP.
Biosecur Bioterror. 2013 Dec;11(4):237-50.
Mouse in vivo neutralization of Escherichia coli Shiga toxin 2 with monoclonal antibodies
Cheng LW, Henderson TD, Patfield S, Stanker LH, He X.
Toxins (Basel). 2013 Oct 22;5(10):1845-58.
A Polyclonal Antibody Based Immunoassay Detects Seven Subtypes of Shiga Toxin 2 Produced by Escherichia coli in Human and Environmental Samples
He X, Patfield S, Hnasko R, Rasooly R, Mandrell RE.
PLoS One. 2013 Oct 16;8(10):e76368.
Development of Monoclonal Antibodies and Immunoassays for Sensitive and Specific Detection of Shiga Toxin Stx2f
Skinner C, Patfield S, Stanker L, He X.
PLoS One. 2013 Sep 17;8(9):e76563.
Non-linear relationships between aflatoxin B1 levels and the biological response of monkey kidney vero cells
Rasooly R, Hernlem B, He X, Friedman M.
Toxins (Basel). 2013 Aug 14;5(8):1447-61.
Low levels of aflatoxin b1, ricin, and milk enhance recombinant protein production in Mammalian cells
Rasooly R, Hernlem B, Friedman M.
PLoS One. 2013 Aug 5;8(8):e71682.
Substrates and controls for the quantitative detection of active botulinum neurotoxin in protease-containing samples
Bagramyan K, Kaplan BE, Cheng LW, Strotmeier J, Rummel A, Kalkum M.
Anal Chem. 2013 Jun 4;85(11):5569-76.
Detoxification of castor meal through reactive seed crushing
Dubois J, Piccirilli A, Magne J, He X.
Ind Crop Prod. 2013 May;43:194-9.
Purification and characterization of neurotoxin complex from a dual toxin gene containing Clostridium botulinum strain PS-5
Singh AK, Sachdeva A, Degrasse JA, Croley TR, Stanker LH, Hodge D, Sharma SK .
Protein J. 2013 Apr;32(4):288-96.
Purification and characterization of Shiga toxin 2f, an immunologically unrelated subtype of Shiga toxin 2
Skinner C, McMahon S, Rasooly R, Carter JM, He X.
PLoS One. 2013 Mar 26;8(3):e59760.
Development and characterization of monoclonal antibodies against Shiga toxin 2 and their application for toxin detection in milk
He X, McMahon S, Skinner C, Merrill P, Scotcher MC, Stanker LH.
J Immunol Methods. 2013 Mar;389(1):18-28.
Development and characterization of six monoclonal antibodies to hemagglutinin-70 of clostridium botulinum and their application in a sandwich ELISA
Scotcher MC, Cheng LW, Ching K, McGarvey J, Hnasko R, Stanker L.
Monoclon Antib Immunodiagn Immunother. 2013 Feb 20;32(1):6-15.
A 7-plex microbead-based immunoassay for serotyping Shiga toxin-producing Escherichia coli
Clotilde LM, Bernard C 4th, Salvador A, Lin A, Lauzon CR, Muldoon M, Xu Y, Lindpaintner K, Carter JM.
J Microbiol Methods. 2013 Feb;92(2):226-30.
Botulinum neurotoxin: where are we with detection technologies?
Singh AK, Stanker LH, Sharma SK.
Crit Rev Microbiol. 2013 Feb;39(1):43-56.
Detection of botulinum neurotoxin serotypes A and B using a chemiluminescent versus electrochemiluminescent immunoassay in food and serum
Cheng LW, Stanker LH.
J Agric Food Chem. 2013 Jan 23;61(3):755-60.
A monoclonal antibody based capture ELISA for botulinum neurotoxin serotype B: toxin detection in Food
Stanker LH, Scotcher MC, Cheng L, Ching K, McGarvey J, Hodge D, Hnasko R.
Toxins. 2013;5(11):2212-26.
Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity
Lee K, Gu S, Jin L, Le TT, Cheng LW, Strotmeier J, Kruel AM, Yao G, Perry K, Rummel A, Jin R.
PLoS Pathog. 2013;9(10):e1003690.
Detection of botulinum neurotoxin serotype A, B, and F proteolytic activity in complex matrices with picomolar to femtomolar sensitivity
Dunning FM, Ruge DR, Piazza TM, Stanker LH, Zeytin FN, Tucker WC.
Appl Environ Microbiol. 2012 Nov;78(21):7687-97.
Milk inhibits the biological activity of ricin
Rasooly R, He X, Friedman M.
J Biol Chem. 2012 Aug 10;287(33):27924-9.
Dynex: multiplex ELISA technology
Clotilde LM, Bernard C 4th, Sequera DE, Karmali A, Fusellier A, Carter JM.
J Lab Autom. 2012 Aug;17(4):309-14.
A single-step purification and molecular characterization of functional Shiga toxin 2 variants from pathogenic Escherichia coli
He X, QuiƱones B, McMahon S, Mandrell RE.
Toxins (Basel). 2012 Jul;4(7):487-504.
Sensitive bioassay for detection of biologically active ricin in food
Rasooly R, He X.
J Food Prot. 2012 May;75(5):951-4.
Detection of ricin contamination in liquid egg by electrochemiluminescence immunosorbent assay
Brandon DL, Korn AM, Yang LL.
J Food Sci. 2012 Apr;77(4):T83-8.
CD154 as a potential early molecular biomarker for rapid quantification analysis of active Staphylococcus enterotoxin A
Rasooly R, Hernlem BJ.
FEMS Immunol Med Microbiol. 2012 Mar;64(2):169-74.
Evaluation and comparison of three enzyme-linked immunosorbent assay formats for the detection of ricin in milk and serum
He X, McMahon S, Rasooly R.
Biocatal Agric Biotechnol. 2012 Jan 9;1(2):105-9.
TNF as biomarker for rapid quantification of active Staphylococcus enterotoxin A in food
Rasooly R, Hernlem B.
Sensors (Basel). 2012;12(5):5978-85.
Electrochemiluminescence immunosorbent assay of ricin in ground beef: Biotinylated capture antibodies and matrix effects
Brandon, DL.
Food Agric Immunol. 2011 Oct 26;23(4):329-37.
Rapid O serogroup identification of the ten most clinically relevant STECs by Luminex microbead-based suspension array
Lin A, Nguyen L, Lee T, Clotilde LM, Kase JA, Son I, Carter JM, Lauzon CR.
J Microbiol Methods. 2011 Oct;87(1):105-10.
Non-Journal Publications:
Risk of ricin from commercial castor production in North America - (Book / Chapter)
Auld, D.L., Trostle, C.L., Miller, T.D., He, X., Duncan, R.W. 2014. Risk of ricin from commercial castor production in North America. Cherwonogrodky, J.W., editor. Ricin Toxin. Oak Park, Illinois:Bentham Science. p. 86-97.
Current technologies for detection of ricin in different matrices - (Book / Chapter)
He, X. 2014. Current technologies for detection of ricin in different matrices.Cherwonogrodky, J.W., editor. Ricin Toxin. Oak Park, Illinois:Bentham Science. p. 38-56.
Detection of bacterial toxins by lateral flow immunoassay - (Proceedings)
Ching, K.H., Stanker, L.H., He, X., Hnasko, R.M. 2013. Detection of bacterial toxins by lateral flow immunoassay. Meeting Proceedings. 6:1-28.
Development of an automated multiplexed immunomagnetic separation system for isolating Shiga toxin-producing Escherichia coli - (Abstract Only)
Accepted Publication (20-Mar-13)
Translocation of botulinum neurotoxins and associated proteins across intestinal epithelial cells(Abstract) - (Abstract Only)
Accepted Publication (16-Mar-13)
Monoclonal antibodies and reagents for botulinum research - (Proceedings)
Stanker, L.H., Cheng, L.W. 2012. Monoclonal antibodies and reagents for botulinum research. The Botulinum Journal. 2(2):150-155 doi: 10.1504/tbj2012.050197.
Replacement of animals in assays for detection of toxins in food(Abstract) - (Abstract Only)
Accepted Publication (10-Jul-12)
Techniques for rapid detection and quantification of active foodborne Staphylococcus Enterotoxin(Abstract) - (Abstract Only)
Hernlem, B.J., Rasooly, R. 2012. Techniques for rapid detection and quantification of active foodborne Staphylococcus Enterotoxin(Abstract). Meeting Abstract. Poster no. B266.
Development of biphasic medium for detection of Shiga toxin producing E. coli using Tetrahymena thermophila - (Abstract Only)
Accepted Publication (17-Feb-12)
Microbead based assays for detection and characterization of E. coli in pure cultures - (Abstract Only)
Accepted Publication (17-Feb-12)
Detection and isolation of non-O157 STECs - (Abstract Only)
Accepted Publication (17-Feb-12)
Detection of botulinum neurotoxin serotypes A and B using chemiluminescence and electrochemiluninescene immunoassays in food and serum matrices - (Abstract Only)
Accepted Publication (21-Dec-11)
Development of a faster method for detection of Shiga toxin producing E. coli using Tetrahymena thermophila - (Abstract Only)
Accepted Publication (15-Oct-11)
Dynex: macrobead multiplex ELISA for STEC - (Abstract Only)
Accepted Publication (15-Oct-11)
Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A - (Abstract Only)
Accepted Publication (07-Oct-11)
Current methods for detecting the presence of botulinum neurotoxins in food and other biological samples - (Book / Chapter)
Cheng, L.W., Land, K.M., Stanker, L.H. 2012. Current methods for detecting the presence of botulinum neurotoxins in food and other biological samples. Morse, S., editor. Bioterrorism. Rijeka, Croatia: Intech. p. 1-16.
Food compounds inhibit Staphylococcus aureus bacteria and the toxicity of Staphylococcus Enterotoxin A (SEA) associated with atopic dermatitis - (Book / Chapter)
Rasooly, R., Friedman, M. 2012. Food compounds inhibit Staphylococcus aureus bacteria and the toxicity of Staphylococcus Enterotoxin A (SEA) associated with atopic dermatitis. In: Esparza-Gordillo, J., editor. Atopic Dermatitis-Disease Etiology and Clinical Management. Croatia: Intech Europe. p. 387-404
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