Quantification of Hordeins by ELISA: The Correct Standard Makes a Magnitude of Difference

Tanner, Gregory J.; Blundell, Malcolm J.; Colgrave, Michelle L.; Howitt, Crispin A.
February 2013
PLoS ONE;Feb2013, Vol. 8 Issue 2, p1
Academic Journal
Background: Coeliacs require a life-long gluten-free diet supported by accurate measurement of gluten (hordein) in gluten-free food. The gluten-free food industry, with a value in excess of $6 billion in 2011, currently depends on two ELISA protocols calibrated against standards that may not be representative of the sample being assayed. Aim: The factors affecting the accuracy of ELISA analysis of hordeins in beer were examined. Results: A simple alcohol-dithiothreitol extraction protocol successfully extracts the majority of hordeins from barley flour and malt. Primary hordein standards were purified by FPLC. ELISA detected different classes of purified hordeins with vastly different sensitivity. The dissociation constant (Kd) for a given ELISA reaction with different hordeins varied by three orders of magnitude. The Kd of the same hordein determined by ELISA using different antibodies varied by up to two orders of magnitude. The choice of either ELISA kit or hordein standard may bias the results and confound interpretation. Conclusions: Accurate determination of hordein requires that the hordein standard used to calibrate the ELISA reaction be identical in composition to the hordeins present in the test substance. In practice it is not feasible to isolate a representative hordein standard from each test food. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes which may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique enabling the quantification of individual hordein isoforms.


Related Articles

  • Insights in the laboratory diagnosis of celiac disease. Basso, D.; Guariso, G.; Fogar, P.; Navaglia, F.; Zambon, C.-F.; Plebani, Mario // Lupus;2006, Vol. 15 Issue 7, p462 

    The present report focuses on the diagnosis of celiac disease and its pathogenesis, which depends on a genetic predisposition (HLA DQ2 or DQ8 haplotypes), gluten ingestion and T cell activation, type II transglutaminase (TG2), the autoantigen recognized by the antiendomysial antibody playing a...

  • Prevalence of celiac disease in patients with autoimmune thyroid disease. Sharma, Bharat; Varthakavi, P. K.; Chadha, M.; Bhagwat, N.; Joshi, A. S.; Lathia, T. B.; Mittal, S.; Sheikh, S.; Dholye, J.; Rahate, S. // Indian Journal of Endocrinology & Metabolism;Dec2012 Supplement, Vol. 16, pS516 

    Introduction: The prevalence of autoimmune thyroid disease (AITD) in India is 7 - 8% and the association of other autoimmune disorders with AITD is well documented. The concomitance of celiac disease (CD) with AITD results in poor absorption of thyroid medications and results in higher doses of...

  • Identification and In Vitro Reactivity of Celiac Immunoactive Peptides in an Apparent Gluten-Free Beer. Real, Ana; Comino, Isabel; Moreno, Mª de Lourdes; López-Casado, Miguel Ángel; Lorite, Pedro; Torres, Mª Isabel; Cebolla, Ángel; Sousa, Carolina // PLoS ONE;Jun2014, Vol. 9 Issue 6, p1 

    Gluten content from barley, rye, wheat and in certain oat varieties, must be avoid in individuals with celiac disease. In most of the Western countries, the level of gluten content in food to be considered as gluten-free products is below 20 parts per million measured by ELISA based on specific...

  • Measuring Hordein (Gluten) in Beer – A Comparison of ELISA and Mass Spectrometry. Tanner, Gregory J.; Colgrave, Michelle L.; Blundell, Malcolm J.; Goswami, Hareshwar P.; Howitt, Crispin A. // PLoS ONE;Feb2013, Vol. 8 Issue 2, p1 

    Background: Subjects suffering from coeliac disease, gluten allergy/intolerance must adopt a lifelong avoidance of gluten. Beer contains trace levels of hordeins (gluten) which are too high to be safely consumed by most coeliacs. Accurate measurement of trace hordeins by ELISA is problematic. ...

  • Cross-Reaction between Gliadin and Different Food and Tissue Antigens. Vojdani, Aristo; Tarash, Igal // Food & Nutrition Sciences;Jan2013, Vol. 4 Issue 1, p20 

    A subgroup of coeliac disease patients continues to experience symptoms even on a gluten-free diet (GFD). We attempted to determine whether these symptoms could be due to either cross-contamination with gluten-containing foods or cross-reactivity between α-gliadin and non-gluten foods...

  • A Non-Human Primate Model for Gluten Sensitivity. Bethune, Michael T.; Borda, Juan T.; Ribka, Erin; Liu, Michael-Xun; Phillippi-Falkenstein, Kathrine; Jandacek, Ronald J.; Doxiadis, Gaby G. M.; Gray, Gary M.; Khosla, Chaitan; Sestak, Karol // PLoS ONE;2008, Vol. 3 Issue 2, p1 

    Background and Aims: Gluten sensitivity is widespread among humans. For example, in celiac disease patients, an inflammatory response to dietary gluten leads to enteropathy, malabsorption, circulating antibodies against gluten and transglutaminase 2, and clinical symptoms such as diarrhea. There...

  • Non-invasive monitoring of celiac disease. Lakos, Gabriella // MLO: Medical Laboratory Observer;Dec2012, Vol. 44 Issue 12, p15 

    The article discusses Celiac disease (CD) and the non-invasive monitoring of CD. It discusses on the non-invasive follow-up and monitoring of CD through serological testing. It discusses about the gluten free diet (GFD) and states that the CD treatment including GFD has now more food options...

  • Investigation of incurred single- and multi-component model food matrices for determination of food proteins triggering allergy and coeliac disease. Török, Kitti; Horváth, Vanda; Horváth, Ágnes; Hajas, Lívia; Bugyi, Zsuzsanna; Tömösközi, Sándor // European Food Research & Technology;Dec2014, Vol. 239 Issue 6, p923 

    For detection and quantification of food proteins triggering hypersensitivity reactions, reliable, validated analytical methods are necessary. At present, the most commonly used methodology for this purpose is ELISA. The lack of incurred reference materials (RM) complicates the fulfilling of...

  • A limp in a pregnant woman as a first presentation of celiac disease. Jammah, A. // Osteoporosis International;Mar2012, Vol. 23 Issue 3, p1183 

    A 25-year-old Arab woman, reported to our endocrinology clinic one month post-partum presenting with back pain and a limp that started during the seventh month of pregnancy. Upon examination, she was found to have a full range of motion and no tenderness in the hip joint or lower back. The pain...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics