Anti-idiotypic nanobody-phage based real-time immuno-PCR for detection of hepatocarcinogen aflatoxin in grains and feedstuffs.

Aflatoxins are a group of extremely toxic small molecules that have been involved in human hepatic and extrahepatic carcinogenesis as causative agents. Herein, we developed a real-time immuno polymerase chain reaction (IPCR) assay for the accurately quantitative detection of aflatoxins in agri-products base on a M13 phage containing aflatoxin anti-idiotypic nanobody and its encoding DNA which was used to design the specific primers. The limit of detection (LOD) of the assay is 0.02 ng/mL, which exhibits a 4-fold improvement over traditional phage ELISA. The developed method was successfully validated with the samples of corn, rice, peanut, and feedstuff, which are major aflatoxin-contaminated agri-products. And the recoveries were from 77.05 to 122.16%. For further validation, the developed assay was also compared with a reference HPLC method for the analysis of aflatoxins in corn and peanuts, and concordant results (R(2) = 0.991) were obtained. In this context, this study provides a novel opportunity to analyze aflatoxins in agri-products.

[1]  B. Hammock,et al.  Isolation of alpaca anti-idiotypic heavy-chain single-domain antibody for the aflatoxin immunoassay. , 2013, Analytical chemistry.

[2]  G. Shephard,et al.  Aflatoxin analysis at the beginning of the twenty-first century , 2009, Analytical and bioanalytical chemistry.

[3]  S. Eremin,et al.  Direct competitive ELISA based on a monoclonal antibody for detection of aflatoxin B1. Stabilization of ELISA kit components and application to grain samples , 2006, Analytical and Bioanalytical Chemistry.

[4]  P. Quesenberry,et al.  A Specific Heptapeptide from a Phage Display Peptide Library Homes to Bone Marrow and Binds to Primitive Hematopoietic Stem Cells , 2004, Stem cells.

[5]  S. M. Herzallah,et al.  Determination of aflatoxins in eggs, milk, meat and meat products using HPLC fluorescent and UV detectors , 2009 .

[6]  Daohong Zhang,et al.  Ultrasensitive nanogold probe-based immunochromatographic assay for simultaneous detection of total aflatoxins in peanuts. , 2011, Biosensors & bioelectronics.

[7]  J. Kyd,et al.  Matrix effect and cross-reactivity of select amphetamine-type substances, designer analogues, and putrefactive amines using the Bio-Quant direct ELISA presumptive assays for amphetamine and methamphetamine. , 2007, Journal of analytical toxicology.

[8]  Chang Ming Li,et al.  A simple strategy to obtain ultra-sensitive single-chain fragment variable antibodies for aflatoxin detection , 2013 .

[9]  R. Joerger,et al.  High sensitivity multianalyte immunoassay using covalent DNA-labeled antibodies and polymerase chain reaction. , 1995, Nucleic acids research.

[10]  C. V. Van Peteghem,et al.  Development of an immunoassay-based lateral flow dipstick for the rapid detection of aflatoxin B1 in pig feed. , 2005, Journal of agricultural and food chemistry.

[11]  Peiwu Li,et al.  IMMUNOASSAYS FOR AFLATOXINS , 2009 .

[12]  Achille Cappiello,et al.  Determination of aflatoxins in peanut meal by LC/MS with a particle beam interface , 1995 .

[13]  J. Groopman,et al.  Aflatoxin: a 50-year odyssey of mechanistic and translational toxicology. , 2011, Toxicological sciences : an official journal of the Society of Toxicology.

[14]  T. Chiba,et al.  Isolation and characterization of phage-displayed single chain antibodies recognizing nonreducing terminal mannose residues. 1. A new strategy for generation of anti-carbohydrate antibodies. , 2007, Biochemistry.

[15]  R. Niessner,et al.  Use of isotope-labeled aflatoxins for LC-MS/MS stable isotope dilution analysis of foods. , 2008, Journal of agricultural and food chemistry.

[16]  Y. Ando,et al.  Biosynthetic relationship among aflatoxins B1, B2, G1, and G2 , 1988, Applied and environmental microbiology.

[17]  Zhaowei Zhang,et al.  Advanced hyphenated chromatographic-mass spectrometry in mycotoxin determination: current status and prospects. , 2013, Mass spectrometry reviews.

[18]  I. Kennedy,et al.  A rapid aflatoxin B1 ELISA: development and validation with reduced matrix effects for peanuts, corn, pistachio, and Soybeans. , 2004, Journal of agricultural and food chemistry.

[19]  Daohong Zhang,et al.  Production of ultrasensitive generic monoclonal antibodies against major aflatoxins using a modified two-step screening procedure. , 2009, Analytica chimica acta.

[20]  P Delfosse,et al.  Production and characterization of monoclonal antibodies for aflatoxin B1 , 1999, Letters in applied microbiology.

[21]  Manju Sharma,et al.  Determination of aflatoxins in domestic pet foods (dog and cat) using immunoaffinity column and HPLC , 2001 .

[22]  L. Ravarotto,et al.  Efficacy of silymarin-phospholipid complex in reducing the toxicity of aflatoxin B1 in broiler chicks. , 2004, Poultry science.

[23]  Andrea Bolognesi,et al.  Detection of ricin and other ribosome-inactivating proteins by an immuno-polymerase chain reaction assay. , 2006, Analytical biochemistry.

[24]  R. Casagrande,et al.  An outbreak of aflatoxin poisoning in dogs associated with aflatoxin B1–contaminated maize products , 2013, Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc.

[25]  Wenbin Liu,et al.  A Novel Peptide Isolated from a Phage Display Peptide Library with Trastuzumab Can Mimic Antigen Epitope of HER-2* , 2005, Journal of Biological Chemistry.

[26]  Mark R. McCoy,et al.  Noncompetitive phage anti-immunocomplex real-time polymerase chain reaction for sensitive detection of small molecules. , 2011, Analytical chemistry.

[27]  G. P. Smith,et al.  Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. , 1985, Science.

[28]  L. Wyns,et al.  Camelid immunoglobulins and nanobody technology. , 2009, Veterinary immunology and immunopathology.

[29]  B. Tey,et al.  Detection of hepatitis B virus core antigen by phage display mediated TaqMan real-time immuno-PCR. , 2013, Journal of virological methods.

[30]  G. Pulina,et al.  Transfer of aflatoxin B1 from feed to milk and from milk to curd and whey in dairy sheep fed artificially contaminated concentrates. , 2005, Journal of dairy science.

[31]  Hwan-Wun Liu,et al.  A highly sensitive immuno-polymerase chain reaction assay for Clostridium botulinum neurotoxin type A. , 2004, Toxicon : official journal of the International Society on Toxinology.

[32]  J. Verhoef,et al.  Rapid detection of salmonellae in poultry with the magnetic immuno-polymerase chain reaction assay , 1993, Applied and environmental microbiology.