Simultaneous quantification of five bacterial and plant toxins from complex matrices using a multiplexed fluorescent magnetic suspension assay.

Proteotoxins such as ricin, abrin, botulinum neurotoxins type A and B (BoNT/A, BoNT/B) and staphylococcal enterotoxin B (SEB) are regarded as potential biological warfare agents which could be used for bioterrorism attacks on the food chain. In this study we used a novel immunisation strategy to generate high-affinity monoclonal and polyclonal antibodies against native ricin, BoNT/A, and BoNT/B. The antibodies were used along with antibodies against SEB and abrin to establish a highly sensitive magnetic and fluorescent multiplex bead array with excellent sensitivities between 2 ng/L and 546 ng/L from a minimal sample volume of 50 microL. The assay was validated using 20 different related analytes and the assay precision was determined. Advancing the existing bead array technology, the novel magnetic and fluorescent microbeads proved amenable to enrichment procedures, by further increasing sensitivity to 0.3-85 ng/L, starting from a sample volume of 500 microL. Furthermore, the method was successfully applied for the simultaneous identification of the target toxins spiked into complex food matrices like milk, baby food and yoghurt. On the basis of our results, the assay appears to be a good tool for large-scale screening of samples from the food supply chain.

[1]  G. Anderson,et al.  Multi-analyte interrogation using the fiber optic biosensor. , 2000, Biosensors & bioelectronics.

[2]  L. Tauc,et al.  Antagonism of the intracellular action of botulinum neurotoxin type A with monoclonal antibodies that map to light-chain epitopes. , 1994, European journal of biochemistry.

[3]  T. Thomas,et al.  Purification and physicochemical properties of ricins and agglutinins from Ricinus communis. , 1980, European journal of biochemistry.

[4]  K. Rock,et al.  Efficient major histocompatibility complex class I presentation of exogenous antigen upon phagocytosis by macrophages. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Reinhard Niessner,et al.  Automated analytical microarrays: a critical review , 2008, Analytical and bioanalytical chemistry.

[6]  A A Boiarski,et al.  The development of immunoassays to four biological threat agents in a bidiffractive grating biosensor. , 2000, Biosensors & bioelectronics.

[7]  N. Mantis,et al.  Characterization of a Novel High-Affinity Monoclonal Immunoglobulin G Antibody against the Ricin B Subunit , 2006, Infection and Immunity.

[8]  D. Swerdlow,et al.  Threat of a biological terrorist attack on the US food supply: the CDC perspective , 2002, The Lancet.

[9]  B. Shen,et al.  A novel neutralizing monoclonal antibody against both ricin toxin A and ricin toxin B, and application of a rapid sandwich enzyme-linked immunosorbent assay. , 2006, Hybridoma.

[10]  S. Moochhala,et al.  Rapid detection of botulinum neurotoxins A, B, E, and F by optical immunoassay. , 2008, Frontiers in bioscience : a journal and virtual library.

[11]  K. Rock,et al.  Analysis of MHC class II presentation of particulate antigens of B lymphocytes. , 1996, Journal of immunology.

[12]  D. Vignali.  Multiplexed particle-based flow cytometric assays. , 2000, Journal of immunological methods.

[13]  E. Harlow,et al.  Using Antibodies: A Laboratory Manual , 1999 .

[14]  Elizabeth K Wheeler,et al.  Magnetic bead based immunoassay for autonomous detection of toxins. , 2008, Analytical chemistry.

[15]  A. Scheffold,et al.  Single-cell analysis of the murine chemokines MIP-1α, MIP-1β, RANTES and ATAC/lymphotactin by flow cytometry , 2003 .

[16]  Stephen W. Farrow,et al.  Autonomous detection of aerosolized biological agents by multiplexed immunoassay with polymerase chain reaction confirmation. , 2005, Analytical chemistry.

[17]  Lei Zhou,et al.  Simultaneous detection of five biothreat agents in powder samples by a multiplexed suspension array , 2009, Immunopharmacology and immunotoxicology.

[18]  G. Anderson,et al.  Amplification of microsphere-based microarrays using catalyzed reporter deposition. , 2008, Biosensors & bioelectronics.

[19]  Philip K. Russell,et al.  Botulinum toxin as a biological weapon: medical and public health management. , 2001, JAMA.

[20]  R. C. Whiting,et al.  Detection of Type A, B, E, and F Clostridium botulinum Neurotoxins in Foods by Using an Amplified Enzyme-Linked Immunosorbent Assay with Digoxigenin-Labeled Antibodies , 2006, Applied and Environmental Microbiology.

[21]  M. Dorner,et al.  Monitoring of laying capacity, immunoglobulin Y concentration, and antibody titer development in chickens immunized with ricin and botulinum toxins over a two-year period , 2009, Poultry science.

[22]  L. Peruski,et al.  Immunological Methods for Detection and Identification of Infectious Disease and Biological Warfare Agents , 2003, Clinical Diagnostic Laboratory Immunology.

[23]  Hannu Korkeala,et al.  Laboratory Diagnostics of Botulism , 2006, Clinical Microbiology Reviews.

[24]  R. Weselake,et al.  A novel biological-based assay for the screening of neutralizing antibodies to ricin. , 1999, Hybridoma.

[25]  T. Scheel,et al.  Development and validation of a multiplex add-on assay for sepsis biomarkers using xMAP technology. , 2006, Clinical chemistry.

[26]  Emily K. Reinke,et al.  High-Affinity, Protective Antibodies to the Binding Domain of Botulinum Neurotoxin Type A , 2001, Infection and Immunity.

[27]  L. Peruski,et al.  Rapid and sensitive detection of biological warfare agents using time-resolved fluorescence assays. , 2002, Journal of immunological methods.

[28]  J. Justin Gooding,et al.  Biosensor technology for detecting biological warfare agents : Recent progress and future trends , 2006 .

[29]  Aravind Chakrapani,et al.  Nanoparticles and microparticles as vaccine-delivery systems , 2007, Expert review of vaccines.

[30]  Douglas L. Park,et al.  Feasibility of immunodiagnostic devices for the detection of ricin, amanitin, and T-2 toxin in food. , 2005, Journal of food protection.

[31]  Frances S. Ligler,et al.  Array biosensor for detection of toxins , 2003, Analytical and bioanalytical chemistry.

[32]  E. Ezan,et al.  Detection of functional ricin by immunoaffinity and liquid chromatography-tandem mass spectrometry. , 2007, Analytical chemistry.

[33]  Jinbiao Zhan,et al.  A simplified method to evaluate the acute toxicity of ricin and ricinus agglutinin. , 2003, Toxicology.

[34]  Gerhard Walzl,et al.  An Evaluation of Commercial Fluorescent Bead-Based Luminex Cytokine Assays , 2008, PloS one.

[35]  E J Schantz,et al.  Properties and use of botulinum toxin and other microbial neurotoxins in medicine. , 1992, Microbiological reviews.

[36]  K. Rock,et al.  Fully mobilizing host defense: Building better vaccines , 1998, Nature Biotechnology.

[37]  K. Rock,et al.  Cloned dendritic cells can present exogenous antigens on both MHC class I and class II molecules. , 1997, Journal of immunology.

[38]  C. Harding,et al.  Phagocytic processing of exogenous particulate antigens by macrophages for presentation by class I MHC molecules. , 1994, Journal of immunology.

[39]  K. Cole,et al.  Fluorescent nanometer microspheres as a reporter for sensitive detection of simulants of biological threats using multiplexed suspension arrays. , 2005, Bioconjugate chemistry.

[40]  P. Thullier,et al.  Comparison of an electrochemiluminescence assay in plate format over a colorimetric ELISA, for the detection of ricin B chain (RCA-B). , 2007, Journal of immunological methods.

[41]  Leonard A. Smith,et al.  Protective immunity against botulism provided by a single dose vaccination with an adenovirus-vectored vaccine. , 2007, Vaccine.

[42]  E. Bailey,et al.  Toxins of concern to animals and people. , 2006, Revue scientifique et technique.

[43]  S. Ramakumar,et al.  Structure-Function Analysis and Insights into the Reduced Toxicity of Abrus precatorius Agglutinin I in Relation to Abrin* , 2006, Journal of Biological Chemistry.

[44]  Valérie Gros,et al.  Development and comparison of two immunoassay formats for rapid detection of botulinum neurotoxin type A. , 2007, Journal of immunological methods.

[45]  K. Knudsen.  Proteins transferred to nitrocellulose for use as immunogens. , 1985, Analytical biochemistry.

[46]  M. Kimura,et al.  Conserved amino acid residues in ribosome-inactivating proteins from plants. , 1991, Biochimie.

[47]  J. Brown,et al.  Sensitive enzyme-linked immunosorbent assay for detection of Clostridium botulinum neurotoxins A, B, and E using signal amplification via enzyme-linked coagulation assay , 1993, Journal of clinical microbiology.

[48]  C. Shone,et al.  Monoclonal antibody-based immunoassay for type A Clostridium botulinum toxin is comparable to the mouse bioassay , 1985, Applied and environmental microbiology.

[49]  D. Ford,et al.  Experimental botulism in monkeys--a clinical pathological study. , 1967, Experimental and molecular pathology.

[50]  Mary T McBride,et al.  Multiplexed liquid arrays for simultaneous detection of simulants of biological warfare agents. , 2003, Analytical chemistry.

[51]  Lisa C Shriver-Lake,et al.  The Array Biosensor: Portable, Automated Systems , 2007, Analytical sciences : the international journal of the Japan Society for Analytical Chemistry.

[52]  Susan Budavari,et al.  The Merck index : an encyclopedia of chemicals, drugs, and biologicals , 1983 .