Published on October 4, 2007
Detection of Cereal Proteins and DNA Using MS, ELISA, and PCR: Detection of Cereal Proteins and DNA Using MS, ELISA, and PCR William J. Hurkman, Ph. D. USDA/ARS Western Regional Research Center Albany, CA Slide2: Wheat Flour Proteins Slide3: Gliadins and Glutenins Are Prolamins Barley, rye, and oat have proteins that are similar to wheat gliadins. Slide4: Mass Spectrometry (MS) Monoclonal Antibodies (ELISA) Polymerase Chain Reaction (PCR) Several analytical tools are currently available to detect the presence of cereal prolamins in food products: Detection Methods Slide5: Endosperm proteins Spot Matching, Quantification, Profiling 2-DE Gel Spots Wheat Endosperm Protein Database O-MALDI MS: Peptide mass mapping Tandem MS: Peptide sequencing Digest Pro Tryptic fragments MS Protein Identification Sciex QSTAR Data Wheat Grain Slide6: Wheat Endosperm Proteins Peroxidase SERPIN GAPDH Slide7: Many proteins can be separated simultaneously. Protein identification is relatively rapid. Proteins can be quantified. Proteins useful for antibody production. Limited to most abundant proteins. Technically demanding. Equipment is expensive. Pros Cons MS Protein Identification ELISA: ELISA Takes advantage of the ability of antibodies to recognize and bind to proteins (antigens). More rapid and less expensive than mass spectrometry. Highly specific through use of monoclonal antibodies. (Enzyme-Linked ImmunoSorbant Assay) Slide9: Monoclonal Antibody Production Slide10: Sandwich ELISA Sample & Stds Conjugate Substrate Inc. Inc. Inc. Wash Wash Stopping Reagent + - or Slide11: Detects: Wheat gliadins Barley hordeins Rye secalins But not in oat avenins. R5 Monoclonal Antibody Méndez et al. Eur. J. Gastroenterol. (2003) 15:465. Slide12: MKTFLIFVLLAMAMKIATAARELNPSNKELQSPQQSFSYQQQPFPQQPYPQQPYPSQQPYPSQQPFPTPQQQFPEQSQQPFTQPQQPTPIQPQQPFPQQPQQPQQPFPQPQQPFPWQPQQPFPQTQQSFPLQPQQPFPQQPQQPFPQPQLPFPQQSEQIIPQQLQQPFPLQPQQPFPQQPQQPFPQPQQPIPVQPQQSFPQQSQQSQQPFAQPQQLFPELQQPIPQQPQQPFPLQPQQPFPQQPQQPFPQQPQQSFPQQPQQPYPQQQPYGSSLTSIGGQ R5 Monoclonal Antibody Recognizes QQPFP Repetitive sequence in gliadins, hordeins, and secalins, but not in avenins. glutamine-glutamine-proline-phenylalanine-proline R5 ELISA Pros: R5 ELISA Pros Detects wheat, barley, and rye prolamins. Works well for a variety of unprocessed and heat-processed foods. Relatively rapid (1 h 30 min). Sensitive (1.5 ppm). Available in commercial kits. Tested in 20 laboratories (results soon to be published). Temporarily endorsed by the Committee on Methods of Analysis and Sampling of the Codex Alimentarius Commission (more data needed). R5 ELISA Cons: R5 ELISA Cons Does not distinguish between wheat, barley, and rye. Detects only gliadins in wheat. Celiac patients also react to high and low molecular weight glutenins. Some ingredients have glutenins, but not gliadins. Slide15: PCR (Polymerase Chain Reaction) Amplification Denature Template DNA Anneal Primers (Forward & Reverse Oligonucleotides) Extend DNA (DNA polymerase, DNTPs) Slide16: PCR Primers for Prolamins Sandberg et al. Eur. Food Res. Technol. (2003) 217: 344-349. Slide17: PCR Primers for Prolamins Sandberg et al. Eur. Food Res. Technol. (2003) 217: 344-349. Slide18: PCR Specificity Primer Pairs Species Wheat Rye Barley Oat Sandberg et al. Eur. Food Res. Technol. (2003) 217: 344-349. PCR: PCR Species specific: distinguishes between wheat, rye, barley, and oats. Sensitive and rapid. Compliments ELISA results. Detects DNA rather than actual protein. Pros Cons Summary: Summary Mass spectrometry Excellent tool for protein identification. Sensitive, but not rapid. Expensive and technically demanding. ELISA Works well for a variety of unprocessed and heat-processed foods. Sensitive and rapid. PCR Species specific. Compliments ELISA. Sensitive and rapid.