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Public Meeting on Gluten-Free Food Labeling - Text Version of PowerPoint Presentation by William J. Hurkman

Public Meeting: Gluten-Free Labeling main page



 

Slide 1 - Detection of Cereal Proteins and DNA Using MS, ELISA, and PCR

William J. Hurkman, Ph. D.
USDA/ARS
Western Regional Research Center Albany, CA

Slide 2 - Wheat Flour Proteins

Image of a flow chart explaining how wheat flour proteins are separated. At the top is an oval with the word "Dough" in the middle. An arrow pointing down is coming out of the bottom of the oval with the words "Wash out starch granules" across the arrow. The arrow points to another oval with the word "Gluten" in the middle of it. An arrow comes out of the bottom of this oval pointing to the words "Mix with alcohol/water." From this phrase there is an arrow pointing down from the left side to an oval labeled "Glutenins." The word Insoluble" is across this arrow. On the right side of the phrase is an arrow pointing to an oval labeled "Gliadins" with "Soluble" written across the arrow. 

Slide 3 - 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

 

Slide 4 - Detection Methods

Several analytical tools are currently available to detect the presence of cereal prolamins in food products:

  • Mass Spectrometry (MS)

  • Monoclonal Antibodies (ELISA)

  • Polymerase Chain Reaction (PCR)

Slide 5 - MS Protein Identification

Photos - lab equipment used for protein identification. A photo of three pieces of wheat grain in the top right corner. An arrow points out of the wheat grain image labeled "Endosperm porteins" points to the right to an image of blue specs on a white background. This image is labeled 2-DE. From this image an arrow labeled "Gel Spots" points to the right to an image of a Digest Pro machine. From this image an arrow points down labeled "Tryptic fragments." The arrow is pointing to an image of a large cabinet-like machine with  the words "O-MALDI MS: Peptide mass mapping Tandem MS Peptide sequencing" under it. From the image an arrow point left labeled "Data." There is an image of a graph under this arrow. The arrow points to a photo of a computer screen with data on it. From the computer screen photo two arrow point down. One to the words "Spot Matching, Quantification, Profiling." An one arrow points to "Wheat Endosperm Portein Database." 

Slide 6 - Wheat Endosperm Proteins

Photos of protein blots. See transcript for presenter explanation. 

Slide 7 - MS Protein Identification

Pros

  • Many proteins can be separated simultaneously.

  • Protein identification is relatively rapid.

  • Proteins can be quantified.

  • Proteins useful for antibody production.

Cons

  • Limited to most abundant proteins.

  • Technically demanding.

  • Equipment is expensive.

Slide 8 - ELISA (Enzyme-Linked ImmunoSorbant Assay)

  • 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.

Slide 9 - Monoclonal Antibody Production

Photo of a diagram explaining monoclonal antibody production. See transcript for presenter explanation. 

Slide 10 - Sandwich ELISA

Photo of a diagram explaining sandwich ELISA. See transcript for presenter explanation. 

Slide 11 - Sandwich ELISA

  • Detects:

    • Wheat gliadins
    • Barley hordeins
    • Rye secalins
  • But not in oat avenins.

Méndez et al. Eur. J. Gastroenterol. (2003) 15:465.

Slide 12 - R5 Monoclonal Antibody Recognizes QQPFP

Repetitive sequence in gliadins, hordeins, and secalins, but not in avenins.

MKTFLIFVLLAMAMKIATAARELNPSNKELQSPQQSFSYQQQPFPQQ
PYPQQPYPSQQPYPSQQPFPTPQQQFPEQSQQPFTQPQQPTPIQPQ
QPFPQQPQQPQQPFPQPQQPFPWQPQQPFPQTQQSFPLQPQQPFP
QQPQQPFPQPQLPFPQQSEQIIPQQLQQPFPLQPQQPFPQQPQQPFP
QPQQPIPVQPQQSFPQQSQQSQQPFAQPQQLFPELQQPIPQQPQQP
FPLQPQQPFPQQPQQPFPQQPQQSFPQQPQQPYPQQQPYGSSLTSIGGQ

glutamine-glutamine-proline-phenylalanine-proline

Slide 13 - 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).

Slide 14 - 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.

Slide 15 - PCR (Polymerase Chain Reaction) Amplification

Denature Template DNA

Anneal Primers(Forward & Reverse Oligonucleotides)

Extend DNA (DNA polymerase,DNTPs)

Slide 16 - PCR Primers for Prolamins

Wheat Gliadin (AF280605m, 2315-2495) ....cagaaa gcgagtggaa agatgaaagc aagccatgcg atgaaaacta taaagacaca tgcaccacca aggccacctt ccatcatcca aacttcacac acctagacca caagcatcaa aggcaagcaa gcagtagtca ccacaaatcc aacatgaaga ccttcctcat ctttgtcctc cttgc.....

Rye Secalin (X60295, 325-505) ....tttttc agaaagcgag ttcaatgatg aatgaatcca taccataaca actataaata cacatgcacc attatagtca ccttccatca tccaaacttc acgcaccaag atcagaaaca tcaattccaa gcaagcatta gtaaccacaa atccaacatg aagaccttcc tcatctttgt cctcg.....

Sandberg et al. Eur. Food Res. Technol. (2003) 217: 344-349.

Slide 17 - PCR Primers for Prolamins

Barley Hordein (X03103, 1618-1763) .......att aattcccaaa ctgaacgact acgtgaaaag acagtcacac catgtttgta catccacccc tttgctcgaa atggcgttct tttgctggac agccgagctt cagaatctgc cgtcaagttc ctgagatcca tccacagatg tcgttcacat tgttcgccat g.........

Oat Avenin (J05486 1097-1200) ......cgct cagtggcttc taagaacact acaagagcta tagtactaca taaataccat cagcgtttag ccgatggacc gatcttgtag cggtgacaaa taaaataaaa ..........

Sandberg et al. Eur. Food Res. Technol. (2003) 217: 344-349.

Slide 18 - PCR Specificity

Species

Primer Pairs - Wheat

Primer Pairs - Rye

Primer Pairs - Barley

Primer Pairs - Oat

Winter Wheat

+

-

-

-

Spring Wheat

+

-

-

-

Durum Wheat

+

-

-

-

Spelt

+

-

-

-

Kamut

+

-

-

-

Triticale

+

-

-

-

Rye

-

+

-

-

Barley

-

-

+

-

Oat

-

-

-

+

Sandberg et al. Eur. Food Res. Technol. (2003) 217: 344-349.

Slide 19 - PCR

Pros

  • Species specific: distinguishes between wheat, rye, barley, and oats.

  • Sensitive and rapid.

  • Compliments ELISA results.

Cons

  • Detects DNA rather than actual protein.

Slide 20 - 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.