The document summarizes a presentation given by Päivi Kanerva at the 11th International Gluten Workshop in Beijing, China in August 2012. The presentation discussed the reliability of measuring residual gluten in gluten-free foods. It noted that reliability can be affected by the variety of gluten-detecting antibodies used, reference materials, and changes to proteins from hydrolysis or deamidation during food processing. The presentation concluded that reliability can be improved by selecting the appropriate reference material and accounting for how different prolamins may react with antibodies and be modified during processing.

1. 11th International Gluten Workshop
Beijing, China
August 12-15, 2012
How reliable are the measurements of residual gluten in
gluten-free foods?
Päivi Kanerva
Department of Food and Environmental Sciences
University of Helsinki, Finland
www.helsinki.fi/yliopisto

2. Content
Introduction
Conditions that affect reliability of gluten measurements:
• Variety of gluten-detecting antibodies
• Different reference materials
• Protein hydrolysis
• Protein deamidation
Conclusions
11th International Gluten Workshop, Beijing, 12-15 Aug 2012
Päivi Kanerva www.helsinki.fi/yliopisto

3. Introduction
Coeliac disease
• Coeliac disease is one of the most common food intolerances in the
world, with prevalence of 1-2 %
• Gluten proteins are known to be an environmental factor causing
coeliac disease
• Gluten proteins of wheat and similar proteins of barley and rye are
all harmful for coeliacs
• Harmful proteins trigger an adverse immunoreaction in people with
coeliac disease leading to destruction of small intestine villi
• Current treatment for coeliac disease is a gluten-free diet
11th International Gluten Workshop, Beijing, 12-15 Aug 2012
Päivi Kanerva www.helsinki.fi/yliopisto

4. Introduction
Measuring gluten content
• Gluten content is measured by immunological ELISA methods.
• Methods are based on prolamin-recognizing antibodies.
• The method currently recommended by Codex is known by
names R5-ELISA or the method of Mendez
Sandwich Competitive
11th International Gluten Workshop, Beijing, 12-15 Aug 2012
Päivi Kanerva www.helsinki.fi/yliopisto

5. Variety of gluten-detecting antibodies
R5 antibody raised against rye secalin (Sorell et al. 1998)
– currently in use
Recognizes prolamins of wheat, barley and rye; reacts mainly with QQPFP
ω-gliadin antibody raised against wheat gliadin (Skerritt&Hill 1990)
– predecessor of R5
Recognizes heat-stable omega-type prolamins
α-gliadin antibody raised against wheat gliadin (Spaenij-Dekking et al. 2004)
Recognizes T-cell stimulatory epitopes of wheat, barley and rye
G12 and A1 antibodies raised against 33mer (Moron et al. 2008)
Recognizes prolamins of wheat, barley and rye, and weak reaction to oat
prolamins; G12 reacts mainly with QPQLPY, A1 www.helsinki.fi/yliopisto
with QLPYPQP

6. Kanerva et al 2011 Agric Food Sci 20:206-216.
11th International Gluten Workshop, Beijing, 12-15 Aug 2012
Päivi Kanerva

7. Different reference materials
• Reference material has a major influence on the quantification.
• Reference material should be similar to the analysed protein;
however, complexicity of prolamins makes it difficult
• Different standards are used today in gluten analysis:
PWG gliadin
other gliadin standards (e.g. Sigma)
Peptide standards (synthetic or hydrolyzed)
11th International Gluten Workshop, Beijing, 12-15 Aug 2012
Päivi Kanerva www.helsinki.fi/yliopisto

8. Measurement of gluten with R5 ELISA
using different reference materials
Gluten content (ppm)
Hordein content (ppm)
Amount of barley flour (%) Amount of barley flour (%)
Gliadin standard Hordein standard

9. Hydrolysis of proteins
• Hydrolysis of proteins occurs during many food
manufacturing processes, e.g. brewing
• Hydrolysis can be enhanced by enzymes: either added or
endogenous
• High amounts of prolamin degrading enzymes is developed
during grain germination
11th International Gluten Workshop, Beijing, 12-15 Aug 2012
Päivi Kanerva www.helsinki.fi/yliopisto

10. •Rye protein hydrolysis during sourdough
process
Sourdough from germinated rye contained
only 240-480 ppm of prolamins, which is
about 0,5% from original amount
Both sandwich and competitive assay gave
similar results

11. Protein deamidation
In addition to hydrolysis, solubility of gluten can be improved by
deamidation
Deamidation improves also foam and emulsion forming cababilities of
gluten proteins
Deamidation can be done by acid or base treatment or enzymatically
Deamidation is a conversion of glutamine or asparagine to
glutamic Gluten Workshop, Beijing, 12-15acid
11th International acid or aspartic Aug 2012
Päivi Kanerva www.helsinki.fi/yliopisto

12. Chemical deamidation of vital gluten
by acid treatment
11th International Gluten Workshop, Beijing, 12-15 Aug 2012
Päivi Kanerva

13. Deamidation of gluten significantly Omega-gliadin sandwich
reduced its immunological detection ELISA
by the prolamin specific antibodies
R5 and omega-gliadin.
R5 competitive ELISA R5 sandwich ELISA
x 600
x 125
Kanerva et al. 2011. J Cereal Sci

14. Conclusions
Reliability of the measurements of gluten content is questioned if the
complexity of prolamins and changes in their structures are not carefully
considered.
 reliability can be substantially improved by selecting right reference
material
 reactivity of different prolamins with different antibodies should be
taken into account and results interpreted accordingly
 consequences of modification to protein reactivity with antibodies
should be evaluated
11th International Gluten Workshop, Beijing, 12-15 Aug 2012
Päivi Kanerva www.helsinki.fi/yliopisto

15. Thank you
Cereal Technology Group at the University of Helsinki
Professor
Hannu Salovaara
University lecturer
Tuula Sontag-Strohm
Researchers
Päivi Kanerva
Xin Huang
Technician
Outi Brinck