Detection of Gluten by Commercial Test Kits - ACS Publications

Chapter 30

Detection of Gluten by Commercial Test Kits: Effects of Food Matrices and Extraction Procedures Downloaded by UNIV OF GUELPH LIBRARY on July 1, 2012 | http://pubs.acs.org Publication Date: October 20, 2008 | doi: 10.1021/bk-2008-1001.ch030

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Tao Geng , Carmen D. Westphal , and Jupiter M . Yeung * 1

Grocery Manufacturers Association, 1350 I Street, NW, Washington, DC 20005 U.S. Food and Drug Administration, 8301 Muirkirk Road, Laurel, MD 20708 Current address: Monsanto Company, North Lindbergh Boulevard, U3H, St. Louis, MO 63167 2

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To investigate the effects of food matrices and extraction procedures for different test kits used for determination of gluten, six commercially available test kits were evaluated: R -Biopharm RIDASCREEN® Gliadin, RIDASCREEN® FAST Gliadin, RIDAQUICK Gliadin, Neogen Veratox® for Gliadin, Tepnel BioKits Gluten Assay kit, and Morinaga Wheat Protein ELISA kit. The food matrices used were gluten-free guar gum and high-fiber bread mix, also spiked with different concentrations of gluten standard reference material (SRM 8418) obtained from National Institute of Standards and Technologies (NIST). In addition, 30 gluten-free samples were surveyed for gluten content. A homogeneity study showed that spiked gluten reference materials were evenly distributed in food matrices. Because sample extraction mixtures used by the kits are different, extraction efficiencies were carefully evaluated, especially for Neogen and R-Biopharm kits, which provide the same two options for sample extraction, aqueous alcohol and cocktail buffer. Results obtained with the six assay systems and extraction procedures differed considerably. Extraction with cocktail gave almost two times higher concentration of gluten than that from ethanol extraction using RIDASCREEN kits. Further investigation of the kits resulted in divergentfindings.Depending on test methods or extraction procedures used, false positives and negatives were observed. © 2008 American Chemical Society In Food Contaminants; Siantar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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This variation creates uncertainty to end users. There is an urgent need for harmonization of the use of reference materials in control standards, and validation of extraction procedures in order to define the analytical results.

Wheat is the staple cereal in many countries and its uses in manufactured foods are ever growing due to the technological qualities of gluten proteins as well as the numerous applications of different wheat fractions. Cereal grains, providing approximately half of the world's supply of human dietary protein, are also used as ingredients in processing aids, such as thickening agents, fillers, and binders, and they can be found in baked goods, processed meat products, confectionery items, and drinks, among many other processed foods. Although wheat is widely consumed, the ingestion and inhalation of wheat proteins may trigger different disorders in sensitive individuals. Wheat allergy is an IgEmediated reaction to wheat proteins, and can manifest as a classical food allergy syndrome affecting skin, gut, respiratory tract, or cardiovascular system (1). In addition, ingestion of wheat proteins may also cause celiac disease, a T-cell mediated intestinal inflammation (2). The different wheat protein fractions involved in the pathogenesis of these distinct disorders are well characterized (3). While albumin is found to be the primary fraction of importance in baker's asthma, gluten protein fractions are toxic to individuals suffering from both wheat allergy and celiac disease. Dietary glutens, the major storage proteins in cereal grains of wheat, rye, and barley, can be potentially toxic to individuals with celiac disease (4). Gluten is a highly heterogeneous group of proteins, and consists of both aqueous alcohol-soluble prolamins and alcohol-insoluble glutelins. Prolamins can be isolated from wheat (gliadins), rye (secalin), barley (hordein), and oats (avenin). Like food allergies, the prevalence of the disease is higher than previously thought, which has become a public health issue (5). It is believed that the prevalence of celiac disease ranges from 1 in 250 ( 0.05.

Results and Discussion Gluten is a highly heterogeneous group of proteins, and consists of prolamins and glutelins that can be isolated from a number of grains such as wheat, corn, rice, rye, barley, and oats (16). But only glutenfromwheat, rye and barley has been associated with reactions of celiac disease (8). Due to the need for having reliable methods to confirm compliance with the requirements for gluten-free labeling, we evaluated six commercial kits (four brands). Table I summarizes the performance characteristics of the commercial test kits on the US market. This information is public or it has been provided by the manufacturers. Not only do the kits differ in antibody specificity but also in the sample extraction procedures. Every kit provides its own sample extraction mixture. Its composition varies from different percentages of aqueous alcohol to buffers containing detergent and reducing agents, or a combination of both in a 2-step extraction. Because the two gluten fractions, prolamins and glutelins,

In Food Contaminants; Siantar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

467 Table II. Homogeneity Study of Spiked High-fiber Bread Mix Using Veratox for Gliadin Gluten Spiked in High-fiber Mix 25 ppm 50 ppm


ft

Mean ± Std.Dev. No. of Tests (ppm) 23.5 db 1.8 4 46.0 ± 4.5

4

P value' 0.27 0.23

One-way ANOVA used to analyze homogeneity. Samples were homogenous when P values were greater than 0.05.

have different solubility properties in these two types of buffer, our evaluation of the kits focused on extraction efficiency in different food matrices.

Spiked Sample Homogeneity The first step in the study was to prepare the samples containing different levels of gluten from high-fiber bread mix spiked with gluten NIST standard SRM 8418 at afinalconcentration 50 ppm; this level was further diluted with the same mix to provide samples containing 25, 10, and 5 ppm gluten. To ensure that the mixing procedure was properly carried out and to avoid variability due to a non-homogeneous sample, four portions of each 50 and 25 ppm gluten samples were analyzed with Veratox Gliadin kit. Results shown in Table II indicate that the samples were homogeneous at the two gluten levels and they could be used in our study.

Determination of Matrix Effect in Blank Samples In this study, two gluten-free samples, guar gum and high-fiber mix bread, in addition to 60% ethanol, were used to investigate the effect of food matrix on the determination of gluten. Whether the model food matrices were extracted by aqueous ethanol or cocktail buffer, no gluten was detected using RIDASCREEN, Neogen or Tepnel kits (Table III). However, Morinaga wheat protein kit showed presumptive positives above the LOQ of 3.13 ppm in both, the gluten-free guar gum (3.3 ppm wheat protein) and the high-fiber bread mix (3.8 ppm wheat protein), but not in matrix-free 60% ethanol. This result must be interpreted with caution. There are two possible explanations for these values. First, the limit of quantitation (LOQ) of die Morinaga kit is the lowest of the six kits evaluated in this study. It is possible that the LOQ has been calculated by manufacturerfromwheat proteins in buffer or food samples differentfromthe ones used in this study, therefore in this situation we could certainly say that



Guar gum Guar gum Bread mix Bread mix Bread mix Bread mix Bread mix 60%EtOH 60%EtOH 60%EtOH 60%EtOH 60% EtOH

Matrix

ND ND ND ND ND ND 16.2 ND ND ND ND 20.8

ND 26.3 ND ND ND ND 32.6 ND ND ND 12.2 30.4 ND 16.4 ND ND ND ND 15.5 ND ND ND ND 17.4 ND 30.4 ND ND ND ND 39.3 ND ND 7.2 13.1 36.3 + + + +

-

+ + + +

-

+

-

(Cut-off 5 ppm)

Rida Quick (LOQ 10 ppm Gluten)

Veratox

ND 41.8 ND ND 13.2 26.0 46.0 ND ND 11.0 23.6 51.0

ND 50.2 ND ND 12.8 22.2 50.8 ND ND 12.4 23.2 45.0

Ethanol Cocktail Ethanol Cocktail Ethanol Ethanol Cocktail Gluten Gluten Gluten Gluten Gluten Gluten Gluten (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm)

(LOQ 5 ppm Gluten)

(LOQ 10 ppm Gluten)

LOQ=limit of quantification; ND=not detected.

0 50 0 5 10 25 50 0 5 10 25 50

Spiked Gluten (ppm)

RIDASCREEN Gliadin

RIDASCREEN Fast Gliadin

ND 55 ND ND ND 21.5 42.5 ND ND 11.2 21.0 39.4

Ethanol Gluten (ppm)

Tepnel (LOQ 10 ppm Gluten)

3.3 54.7 3.8 9.5 17.8 34.2 58.7 ND 5.7 14.7 25.9 52.6

Morinaga (LOQ 3.12 ppm Wheat Protein) Morinaga Buffer Wheat Protein (ppm)

Table III. Effects of Food Matrices, Extraction Procedures and Detection Systems in Determination of Gluten or Wheat Protein Levels in Spiked Samples



469 these positives are due to matrix effects. The second possibility is that the samples are really contaminated with very low levels of gluten, which are not detected by the other kits because they have higher LOQs. The Morinaga kit uses a sample extraction procedure different to those of the other kits. Even though the Morinaga extraction buffer contains a reducing agent and a detergent, like cocktail buffer, they are more concentrated. Moreover, samples are extracted for longer periods of time (overnight up to 12 h) in contrast with the other kits with extraction times up to 100 min (in the case of cocktail). It is unknown if extended extraction times or higher concentrations of the reducing agent and the detergent lead to the extraction of additional proteins or any other component from the matrix responsible for the positive results. A sensitive alternative method like polymerase chain reaction (PCR) or mass spectrometry would be very helpful in deciding whether this positive is due to contamination or matrix effect. Controversial situations, like this one, are examples where the availability of confirmatory methods plays a critical role in the analysis of gluten.

Effects of Extraction Procedures in Gluten Recovery For the evaluation of gluten recovery from food matrices we selected NIST gluten SRM 8418 as reference material because it is available to all, and there is no lot-to-lot variation. Gluten contents of the different spiked samples are summarized in Table III. For the Veratox gliadin kits, all levels of gluten spiked in all three matrices used were detected with accuracy around 100%, even at the LOQ level of 10 ppm in buffer and bread mix. Moreover, no positives were found at 5 ppm, which is below the LOQ of the kit. No differences between ethanol and cocktail were observed. Similarly, Tepnel also detected almost all levels of gluten in all matrices. However, it was not able to detect gluten at the LOQ level of 10 ppm of gluten in bread mix. This is an expected result since we cannot expect to have 100% gluten recovery. Even if the recovery is within the accepted range of 80100% it is very possible that gluten remains undetected in this sample. Three different immunoassays from R-Biopharm were evaluated: two quantitative ELISAs developed in a 96-well microtiter plate format and a third one which is a qualitative lateral flow device, with a cut-off value of 5 ppm gluten that distinguishes between a positive and a negative sample. They use the same monoclonal antibody R5 so similar results are expected. Surprisingly, both RIDASCREEN ELISAs only detected gluten in the 50 ppm samples with about 32% recovery in ethanol extracts versus 70% for the cocktail. Only cocktail extracts showed positives in 60% ethanol spiked with 25 ppm gluten, but recoveries barely reached 50%. Given these results, there are two aspects of recovery that need to be discussed. First, the overall low recovery of gluten from the samples by the two RIDASCREEN ELISAs may not be related to an



470 extraction efficiency problem. We need to be aware that the reference material used by the manufacturer is different from the NIST gluten reference material that we used to spike the food samples in terms of gluten content and gluten proteins. The manufacturer calibrated their control standards against the European reference standard IRMM-480 which is definitely different from NIST SRM 8418. The second aspect of the recovery that needs to be addressed is based on the differences found between the two different sample extraction buffers. In the case of the RIDASCREEN ELISAs, cocktail extracts provided about 2-fold gluten levels compared to ethanol. This observation has been previously reported (17, 18). Garcia et al. (17) found that cocktail yielded gluten levels between 1.1- and 3.0-fold higher than aqueous ethanol in the analysis of unheated products and wheat starch. Different results provided by the same kit with the same sample only create confusion, uncertainty, and questions about the real gluten content in the sample. And more importantly, results like this can create a make-or-break situation in a gluten-free claim regardless of the action levels provided by different regulatory agencies. Both Neogen and R-Biopharm kits provides cocktail or aqueous ethanol as options for sample extraction. Aqueous ethanol has been traditionally used for the extraction of prolamins. However, after the food has been heated, some of the prolamin proteins tend to aggregate, becoming insoluble in this solvent mixture. Cocktail extraction solution contains guanidine hydrochloride, a disaggregating agent, which along with a reducing agent helps solubilize these proteins. At the time of the study, no recommendations were offered as to when alcohol or cocktail should be used. Currently, both Neogen and R-Biopharm recommend cocktail for the analysis of gluten from heated foods such as baked goods. For unprocessed samples, the gluten content determined by the two kits and the two extracts should be similar. Furthermore, even though the resulting gluten content provided by the two kits is different, the ratio of gluten content in cocktail and aqueous ethanol extracts determined by one kit should be the same as that provided by the second kit. However, this is not the case because the cocktail/ethanol ratio for R-Biopharm is 2 while it is 1 for Neogen. The reason for a different ratio is probably due to differences in antibody specificity. RBiopharm uses a single antibody R5 and it has been well characterized and documented. We know that Neogen uses two different monoclonal antibodies (personal communication) but the information about their characterization has not been made public. This type of information is critical to understanding the dynamics of the detection as well as the interpretation of results. Surprisingly, the RidaQuick LFD, which also uses mAb R5, seems to be more sensitive than the ELISA counterpart. It was able to detect gluten in all of the matrices at all levels, including at the 5 ppm cut-off value, but not in the blanks. In our hands, this LFD provides most reliable results for all the samples that we have analyzed, provided that itfitsthe purpose of the testing. Regarding the Morinaga kit, extraction efficiencies reported for all levels in all samples are around or over 100% in most of the cases that could be due to a


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matrix effect as discussed above. Values are very similar to those provided by Neogen. However, similar values do not mean same results because reporting units makes the difference. The Neogen kit reports the results as ppm gluten while the Morinaga kit reports the results as ppm wheat proteins, so even though the numbers are the same they cannot be compared. It is not exactly known what type of conversion factor Morinaga uses to convert ppm of gluten to ppm wheat proteins, if wheat proteins are defined by the manufacturer as the totality of wheat proteins.

Detection of Gluten in Commercial Gluten-free Foods We surveyed 30 commercial gluten-free foods, grouped in more than 8 food categories, as well as four gluten-containing products (Table IV). A l l the analyses were carried out with the Neogen Veratox kit. Among the gluten-free samples, only buckwheat flour had detectable gluten (46.6 ppm). However, four different lots of this gluten-free buckwheat flour were purchased from the same company at different times and no detectable gluten was observed. To better characterize the source of contamination, the sample was analyzed by PCR by an independent lab. The flour was positive for wheat, and negative for rye and barley; oat was not tested by the PCR analysis. The contaminated buckwheat flour sample was subject to further analysis by the six kits (Table V). If the results observed in the previous study with spiked samples can be generalized, we would expect lower values with RBiopharm ELISAs than the rest of the kits. However, results did not follow that pattern. Results for cocktail RIDASCREEN tests are not only higher than the rest, but also are negative for the aqueous ethanol extract, like Tepnel. Neogen also had the same result for both extracts with similar values for Morinaga, analogous to the results from the recovery study. These types of results that do not follow the same pattern are quite disturbing, and there are two possible explanations. One possibility is that the wheat residue is not homogeneously distributed in the bag of buckwheat flour. The second possibility relates to the antibody specificity. We do not know if the antibodies used in these kits have different affinities for gliadins from different wheat cultivars and how different are those to the ones used in control standards. This can certainly cause deviation from the general pattern of results, i.e., that kit 1 always provides twice the result values from those of kit 2. These divergent results are puzzling and make accurate and reliable detection very difficult. This variability will have a bearing on the ability to perform meaningful risk assessments.

Conclusion In this study, six antibody-based gluten detection kits from four companies were evaluated in different matrices and were further used for the determination


472 Table IV. Determination of Gluten Content in Foods with Neogen Veratox


Sample Type

Gluten (ppm) Food Category No. ofSamples ND Mix 5 ND Flour 6 ND/46.6' Buckwheat flour 5 4 ND Sauce Gluten-free 2 ND Noodle ND Bread 2 Cracker ND 2 2 ND Rolls Bagel 2 ND Flour 816 1 Gluten-containing 1817 1 Waffle samples 1809 Cereal 1 1979 Bread 1 * One out of S buckwheat flour packages (same brand, different lots) was found positive. ND=not detected.

of gluten content in gluten-free food samples. Extraction efficiency of extraction mixtures from the kits was carried out in food matrices spiked with NIST gluten reference standard SRM 8418. Results differed from assay to assay. It is difficult to perform a full assessment of the kit performance just with the results provided by the kits. In light of the divergent results, it seems clear that there is a need for standardization of reference standards and sample extraction procedures for the detection of gluten to minimize the potential sources of variability. In fact, almost every aspect of the kits should be carefully evaluated, including sample extraction buffers, type of antibody (monoclonal versus polyclonal), a single antibody versus a combination of them, antibody specificity, reference material, and reporting units. With the information we currently have we do not know which is the best approach or which elements from the different kits are introducing uncertainties and to what extent this uncertainty contributes to the final result. Some of the aspects of these kits can only be clarified by the manufacturers. In order to really compare the kits we need to understand how they were designed, developed, and optimized. Moreover, more research is needed to understand the target analyte(s). In the case of gluten the kits are targeting multiple proteins, which may vary with the type of cultivar. Furthermore, we need a better understanding and characterization of the modifications to the target analyte(s) produced during the production of food. Standardized reference materials suitable for the detection of gluten in food



ND=not detected.

Extraction mixture Target analyte Concentration (ppm) Gluten >80

Ethanol Gluten ND

Cocktail

Gluten

37.5

Gluten

ND

Cocktail

RIDASCREEN Gliadin

Ethanol

RIDASCREEN Fast Gliadin

Positive

Gluten

Ethanol

Rida Quick

46.6

Gluten

Ethanol

49.8

Gluten

Cocktail

Veratox

ND

Gluten

Ethanol

Tepnel

Table V. Gluten Concentrations found in a Gluten-free Buckwheat Flour


59.0

Wheat protein

Buffer

Morinaga

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ingredients and processed foods are desperately needed. For all of these reasons we cannot conclude which kit is the most accurate and the best performer. There is an imperative need for accurate gluten results, which can only be obtained if reliable and validated detection methods are available. The issue has to be addressed with urgency since regulatory agencies are defining the "glutenfree" term and they are proposing rules for its use. "Gluten-free" is a critical action level or threshold that will ensure the health of millions of individuals suffering from celiac disease as well as to maximize their food choices. Clearly, monitoring and risk assessment programs can be jeopardized if the detection issue is not addressed.

References 1.

2. 3.

4. 5.

6. 7.

8. 9.

10. 11. 12. 13. 14.

Scibilia J.; Pastorello, E. A; Zisa, G.; Ottolenghi, A.; Bindslev-Jensen, C.; Pravettoni, V.; Scovena, E.; Robino, A.; Ortolani, C. J. Allergy Clin. Immunol. 2006, 117, 433-439. Periolo, N.; Chernavsky, A. C. Coeliac Dis., Autoimmun. Rev. 2006, 5, 202208. Battais, F.; Richard, C.; Szustakowski, G.; Denery-Papini, S.; MoneretVautrin, D. A.; Leduc, V.; Guerin, L. Allerg. Immunol. (Paris) 2006, 38, 59-61. Marsh, M . N . Gastroenterology 1992, 702, 330-354. Hischenhuber, C.; Crevel, R.; Jarry, B.; Maki, M.; Moneret-Vautrin, D.A.; Romano, A.; Troncone, R.; Ward, R. Aliment. Pharmacol. Ther. 2006, 23, 559-575. Not, T.; Horvath, K.; Hill, I. D.; Partanen, J.; Hammed, A.; Magazzu, G.; Fasano, A. Scand. J. Gastroenterol. 1998, 33, 494-498. Fasano, A.; Berti, I.; Gerarduzzi, T.; Not, T.; Colletti, R. B.; Drago, S.; Elitsur, Y.; Green, P. H. R.; Guandalini, S.; Hill, I. D.; Pietzak, M.; Ventura, A.; Thorpe, M.; Kryszak, D.; Fornaroli, F.; Wasserman, S. S.; Murray, J. A.; Horvath, K. Arch. Intern. Med. 2003, 163, 286-292. McGough, N.; Cummings, J. H. Proc. Nutr. Soc. 2005, 64, 434-450 Arentz-Hansen, H.; Korner, R.; Molberg, O.; Quarsten, H.; Vader, W.; Kooy, Y. M . C.; Lundin, K. E. A.; Koning, F.; Roepstorff, P.; Sollid, L. M.; McAdam, S. N . J. Exp. Med. 2000, 191, 603-612. Sollid, L. M . Annu. Rev. Immunol. 2000, 18, 53-81. Osman, A. A.; Uhlig, H. H.; Valdes, I.; Amin, M.; Mendez, E.; Mothes, T. (2001) Eur. J. Gastroenterol. Hepatol. 2001, 13, 1189-1193. Mendez, E.; Vela, C.; Immer, U.; Janssen, F. W. Eur. J. Gastroenterol. Hepatol. 2005, 17, 1053-1063. Skerritt, J. H.; Hill, A. S. J. Assoc. Off. Anal. Chem. 1991, 74, 257-264. Denery-Papini, S.; Nicolas, Y.; Popineau, Y. J. Cereal Sci. 1999, 30, 121131.


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15. Akiyama, H.; Isuzugawa, K.; Harikai, N . ; Watanabe, H.; Iijima, K.; Yamakawa, H.; Mizuguchi, Y.; Yoshikawa, R.; Yamamoto, M.; Sato, H.; Watai, M.; Arakawa, F.; Ogasawara, T.; Nishihara, R.; Kato, H.; Yamauchi, A.; Takahata, Y.; Morimatsu, F.; Mamegoshi, S.; Muraoka, S.; Honjoh, T.; Watanabe, T.; Sakata, K.; Imamura, T.; Toyoda, M.; Matsuda, R.; Maitani, T. J. Food Hyg. Soc. Jpn. 2004, 45, 128-134. 16. Wiser, H. Food Microbiol. 2007, 24, 115-119.

17. Garcia, E.; Llorente, M.; Hernando, A.; Kieffer, R.; Wieser, H.; Méndez, E. Eur. J. Gastroenterol. Hepatol. 2005, 17, 529-539. Downloaded by UNIV OF GUELPH LIBRARY on July 1, 2012 | http://pubs.acs.org Publication Date: October 20, 2008 | doi: 10.1021/bk-2008-1001.ch030

18. Hasselberg, A.; Kruse, B.; Yman, I. M . In Proceedings of the 18th Meeting of Working Group on Prolamin Analysis and Toxicity; Stern, M . , Ed.;

Verlag Wissenschaftliche Scripten: Zwichau, Germany, 2004; pp 109-118.


Detection of Gluten by Commercial Test Kits - ACS Publications

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