DetectionofCerealProteinsandDNAUsingMS,ELISA,and.pptVIP

Detection of Cereal Proteins and DNA Using MS, ELISA, and PCR William J. Hurkman, Ph. D. USDA/ARS Western Regional Research Center Albany, CA Mass Spectrometry (MS) Monoclonal Antibodies (ELISA) Polymerase Chain Reaction (PCR) Many proteins can be separated simultaneously. Protein identification is relatively rapid. Proteins can be quantified. Proteins useful for antibody production. 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. 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 PCR Species specific: distinguishes between wheat, rye, barley, and oats. Sensitive and rapid. Compliments ELISA results. 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. * * Wheat Flour Proteins Dough Gluten Glutenins Gliadins Wash out starch granules Mix with alcohol/water Soluble Insoluble Gliadins and Glutenins Are Prolamins Barley, rye, and oat have proteins that are similar to wheat gliadins. Cereal Species Name Wheat Triticum Gliadins Barley Hordeum Hordeins Rye Secale Secalins Oat Avena Avenins Several analytical tools are currently available to detect the presence of cereal prolamins in food products: Detection Methods Endosperm proteins Spot Matching, Quantification, Profiling 2-DE Gel Spots Wheat Endosperm Protein