Self-management of dietary compliance in coeliac disease by means of ELISA "home test" to detect gluten

Saturday

16

February

1991

No 8738

ORIGINAL ARTICLES

Self-management of dietary compliance in coeliac disease by means of ELISA "home test" to detect gluten

To improve compliance with a gluten-free diet in coeliac disease a simple prototype test kit was developed to detect gluten in foods for use at home. The test is based on monoclonal antibodies to heat-stable gluten proteins which crossreact appropriately with barley and rye proteins. It is suitable for use with a wide range of raw or cooked foods. The food is extracted with dilute hydrochloric acid and 1 drop of the extract transferred to an

antibody-coated tube; enzyme-labelled gluten detection antibody is added and after 3 min the tube is washed and colour developer is added. The reaction is stopped after 2 min, stabilising the blue colour. The home kit was compared with a quantitative laboratory kit, and the qualitative agreement was very good. The kit could distinguish foods with trace gluten contents (acceptable for a "gluten-free" diet) from those with a slightly higher but unacceptable gluten content. In a trial of the prototype kit by 47 coeliac disease patients of diverse ages and educational backgrounds, 93% of tests correctly identified foods as acceptable or unacceptable. Introduction Coeliac disease (gluten-sensitive enteropathy) is characterised by a permanent hypersensitivity reaction of the small intestine in genetically susceptible individuals after ingestion of gluten proteins from flour or meal of wheat, rye, barley, and possibly oats.1,2It affects between 1 in 300 and 1 in 10 000 people of European origin,1 and several mechanisms of action of gluten on the intestine have been proposed. Symptoms result largely from the intestinal lesion and can include diarrhoea, flatulence, bulky stools, fatigue, and nutritional deficiencies resulting from malabsorption of vitamins, minerals, and aminoacids; in time, these can lead

retarded growth in children and anaemia and bone weaknesses in adults.1,2 The only effective treatment of coeliac disease is strict exclusion of gluten from the diet. The strictness of adherence to the diet can affect the rate and degree of recovery of intestinal morphology and disappearance of the symptoms. It is possible for patients on an incompletely gluten-free diet to present with few or no symptoms, especially teenagers and adults;3,4 however, the risk of some malignant disorders may be greater in those with poor adherence.s A gluten-free diet may be valuable in some patients with arthritis, allergies, and other gut disorders such as Crohn’s disease.6 Maintenance of a gluten-free diet is not as simple as it may seem.7Labelling laws in most countries are imprecise, and added cereal flour or gluten in processed foods is often called "vegetable protein" or "thickener". Food manufacturers often purchase compound flavourings added to foods preformulated, and they may therefore be unsure of the composition. Thus, wheat gluten can be found in soups, processed meals, desserts, in confectionery such as caramels and liquorice, and as a binder of active ingredients in drugs. Wheat starch, if well separated from gluten in its manufacture, is a suitable base for "gluten-free" baking mixes.8 However, quality control of this feature of starch manufacture is difficult, and there have been reports of adverse reactions to supposedly gluten-free foods.9 Ryebased foods are eaten largely in eastern Europe, and barley proteins in a partially hydrolysed form (malt) are used in flavourings and beer. Although the toxicity of oats is unclear, rye and barley should certainly be avoided.lo,l1 Long-established techniques for food protein analysis, such as gel electrophoresis, high-performance liquid chromatography, and microscopy, are slow and can be unreliable after foods have been baked or processed; they are

to

ADDRESS

CSIRO Wheat Research Unit, Division of Plant

Industry, PO Box 7, North Ryde, New South Wales 2113, Australia (J H Skerritt, PhD, A. S. Hill, BSc) Correspondence to Dr J H Skerritt

380

clearly not adaptable to home use by untrained individuals." Tests with polyclonal antisera to gluten provide reliable results only with uncooked products or bakery mixes, and these antibodies may not appropriately detect non-wheat cereals." We raised monoclonal antibodies to heat-stable omega-gliadins in wheat that crossreacted appropriately with rye and barley proteins, which are also toxic in coeliac disease." An enzyme-linked immunosorbent assay (ELISA) test kit based on higher-affinity antibodies has been developed for laboratory use for quantitative analysis of gluten in all types of foods (Cortecs Ltd, Clwyd, UK; Medical Innovations, Labrador, Australia) .14,15 We have now developed a simple test kit to allow rapid semiquantitative or qualitative analysis of gluten in foods that can easily be used in the home.

TABLE I-COMPARISON OF LABORATORY AND HOME TEST KITS FOR DETERMINATION OF GLUTEN IN FOODS

Methods Several gluten solvents were evaluated in terms of crossreaction results with extracts of many cereals, their ability to discriminate an acceptable and just unacceptable wheat starch, and the stability at ambient temperature. Factors such as cost, lack of toxicity, and possibility of providing the extractant in a concentrated form were also important. The solvent used for food extraction in the laboratory tests (ethanol, 40% weight/volume) was satisfactory on most counts, except that liquor laws to not allow use of unadulterated alcohol in home kits, and only a little over two-fold concentrate of the extractant can be made. Other studies have shown that urea16 and dilute acids17 are efficient extractants of the immunoreactive gliadin fraction. Urea (1 mol/1) produced reasonable results with wheat and rye, but barley was poorly detected. Hydrochloric acid (2 mmol/1) gave appropriate crossreactions with extracts of different cereals and good discrimination of acceptable and just unacceptable wheat starches. It was thus chosen for the kit. The peroxidase chromogen system used in the laboratory test,15

2,2’-azinobis (3-ethyl benzthiazoline-6-sulphonic acid) gives a green colour which continues to intensify for about 30 min after substrate addition. Unreacted chromogen is a very pale green colour.

for the home test we chose tetramethylbenzidine,18 since it is quite colourless yet forms a dark blue colour in the presence of peroxidase, so that it was easier to discriminate results for foods containing zero and trace quantities of gluten. Colour development was faster than with the laboratory test

However,

chromogen. In the laboratory test for gluten in foods, gliadins were extracted by use of a high-frequency homogeniser.14 This method extracted 90% of the immunoreactive fraction from a range of foods14,15 but to make the home test method as simple as possible, foods were instead finely chopped and the gluten extracted by shaking for 60 s. For preparation of the test kit polystyrene tubes (Nunc, Denmark) were incubated for 90 min at room temperature (18-23°C) with 1 ml/tube 10 mg/1 monoclonal antibody 401/21,14 diluted in 50 mmol/1 sodium carbonate buffer, pH 9-6. They were then washed three times with phosphate-buffered saline containing 0-05% (weight/volume) ’Tween 20’ and non-specific binding sites were blocked by means of 1 % bovine serum albumin in phosphatebuffered saline for 60 min at room temperature. Alternatively, the antibody may be freeze-dried onto the tubes, and coated tubes stored for up to a year at 4°C or for 6 months at 20°C. Food samples (about 05 g) are extracted with 5 ml 2 mmol/1 hydrochloric acid. Samples can also be measured approximately by volume by means of a line marking 0-5 ml on the extraction tube, and hydrochloric acid added to the second line (5-5 ml). The tube is capped and shaken for 60 s so that the sample is well suspended and most of the gluten dissolves. Before the antibody-coated reaction tubes are used the blocking solution is removed (in the case of freshly prepared tubes), and 0-8 ml bovine serum albumin/tween/ phosphate-buffered saline added (30 drops). 1 drop (30 ul) of food extract is added to the reaction tube, which is capped and the contents mixed for 90 s. 100 pl (3 drops) of horseradish-peroxidaselabelled antibody 401 /21 is added and the tube gently mixed for 90 s. over

*+/- indicates above limit of detection of test (0 016%) but below gluten level found m wheat products on threshold of acceptability for gluten-free labelling under Codex guidelines (WHO/FAO standard 118, 1981, typically 0 020%), + indicates over 0 020%, - indicates below 0 016%. tAfter subtraction of blank value (0 094) tscored by eye by two observers unaware of nature of food sample =pa!er, + / - = equal, or + = greater than results produced by a 0 020% gluten-containing wheat starch (A405=0 0 20) §Samples provided after reports of adverse reactions by several coeliac disease patients ’1 Based on nce and millet

During these steps, gluten components bind to both the antibody immobilised on the tube and enzyme-labelled antibody. Other food components are removed by washing the tube for 20 s under cold tap water. The presence of gluten is detected by addition of substrate 3,3’,5,5’ tetramethylbenzidine (Sigma, St Louis, USA) in 01 mol/I acetate-citrate buffer, pH 5-5, containing 00006% hydrogen peroxide. The substrate turns blue in tubes with foods containing gluten. In some experiments to evaluate the kit, colour development was measured spectrophotometrically at 450 nm after 2 min incubation at room temperature and addition of 2 mol/1 sulphuric acid (0-25 ml) to yield a stable yellow end-product. 47 members of coeliac disease societies in the eastern states of Australia took part in trials of the kits. All had English as their first language and they ranged in age from 7 to 76 years (10-15 years 9%, 16-25 years 9%, 26-45 years 41 %, 46-65 years 34%, 66 years and older 7%). People with a range of educational backgrounds and from both urban and non-urban areas took part. Most of the participants were female (77%), in keeping with the greater female prevalence of coeliac disease in adults, and the greater proportion of women traditionally involved in food preparation in the home. In addition, five dietitians or food technologists tested the kit. Each participant was provided with a kit, instructions, questionnaire, and six food samples as well as a wheat starch sample containing 0 01%

381

TABLE !!—K!TTmAL RESULTS

pale blue colour), borderline gluten content (just unacceptable, pale blue, same as the colour standard provided in the kit), or a high gluten content (producing a colour darker than the kit standard). Although this was the participants’ first exposure to the method and a fairly crude prototype was used (without the benefit of graphic instructions), an average 93% of tests yielded correct a

very

answers.

Discussion

0-09-0-17, less than the result with the borderline standard (0-30). In eight starches with 0-018-0-090% gluten the optical densities were 0-42-0-70, so the test clearly distinguished the two groups of raw materials potentially used for "gluten-free" baking mixes. The performance of the home kit was compared with that of the laboratory kit14,lS on various "gluten-free" specialty foods, raw ingredients for these foods (wheat and maize starches), and other foods (table i). The tests gave very good qualitative agreement. Foods which had trace quantities (below 0-020%) of gluten and which would be acceptable for the majority of coeliac disease patients, gave some colour development in the home test (optical density at 450 nm 011-0 16), which meant that these products could be identified as containing gluten if required. Although the home test was sensitive, unlike the laboratory test it was not designed to distinguish foods that differed in gluten contents but were both unacceptably high for the coeliac diet. Thus, a quite unacceptable wheat starch of 0-09% gluten gave similar results to biscuits and flour of 4-11 % gluten.

The same antibodies and general principles were used for the rapid test as had been developed for the slower (2 h) quantitative laboratory test for gluten in foods.14,15 The important modifications were the use of higher antibody concentrations, small test tubes rather than microwells, a single tap water washing step rather than two buffer washing steps between incubations, and a "higher-turnover" peroxidase chromogen. These modifications contributed to the ease of performance of the test and greatly reduced the time per test. Extraction of gluten from crushed foods by means of brief shaking was necessarily incomplete. This feature and the addition of reagents from dropper bottles made the assay only semiquantitative, but even so it was very sensitive. Most of the trial participants found the method easy to use, despite the lack of step-by-step diagrams for use as would be provided with a commercial version. The rate of correct responses was 82-100%. More importantly, the rate of false negatives was extremely low-at most only 4 subjects found unacceptable samples to be acceptable. The false positive rate was slightly higher, especially for samples that contained gluten at levels below the cutoff value. We intended that these samples would yield a pale blue colour in the test so that the few gluten-allergic or intolerant individuals who are extremely sensitive to gluten can avoid these foods.1920 An acceptable wheat starch was found to be unacceptable by 14% and a (just) acceptable baking mix unacceptable by 18% of participants. Subsequent questioning of these trial participants showed that most tended to allow colour to develop for more than the 2 min required since they had noticed traces of colour developing within that time. Thus, the frequency of false positives could be reduced either by placing greater emphasis on the importance of an exact 2 min colour development step or by provision of a dropper bottle of peroxidase inhibitor in the kit, to stop further colour development after 2 min. Testing for gluten in foods necessarily involves an additional step beyond those in the only antibody based test in widespread use in the home, the home pregnancy test. Whereas pregnancy tests measure a soluble hormone in urine, for gluten testing the antigen must first be extracted into solution from the study food. The development of simple sensitive and specific tests for "lay" individuals (with specific food allergies or intolerances) to use in a home setting, opens up a whole new area of antibody-based testing. Other food analytes that could be tested by means of antibody (or lectin-based) tests include lactose and proteins from eggs, milk, and legumes or contaminants in food, such as pesticides. Coeliac disease is no longer a disorder with high mortality, but the treatment, adherence to a strict gluten-free diet, involves substantial limitations on patients’

The results of the trial of the kit by coeliac disease patients shown in table II. 3 participants who advised us that they had not followed the instructions correctly were not included. The partipants were required to indicate whether the sample had an acceptable gluten content (resulting in

lifestyles.1 Apart from the obvious application of the method to monitoring of "new" packaged processed foods in the home by coeliac disease patients, "gluten-free" products which are established in their diets require checking from time to time

*Responses implying unacceptabllity for borderline and high) tjust acceptable #Unacceptable

a

gluten-free

diet have been

pooled (ie,

gluten (0-29% protein) which was acceptable for "gluten-free" diets. The gluten contents of two of the samples were indicated: a wheat starch containing 0-15% gluten (0-45% protein, unacceptable for gluten-free diets) and a wheaten bread (11% gluten). Four "blind" samples were provided-a cooked processed meat product, two "gluten-free" baking mixes, and a vegetable soup. Different participants received different (coded) food samples. Results With the home test method, extraction of gluten was .

quantitative from a 0-03% gluten starch, but only 62% complete from a 0-1% gluten starch, and 18% complete from a flour with 11 % gluten. Incomplete extraction at high gluten contents was not important since the test did not aim to discriminate gluten contents above 0-08%. In a set of five wheat starches with 0’005-0-016% gluten (acceptable for gluten-free diets) the optical densities produced in the rapid test were

are

382

because of possible gluten contamination due to recipe or raw material changes by manufacturers. Some foods, such as butchers’ sausages, are not labelled, even if they have been manufactured elsewhere. Health professionals, such as dietitians, could use the test to check hospital diets and to check the composition of drugs and other medicines for

gluten-intolerant patients. Consumption of exotic and ethnic foods is increasingly popular in western society, but difficulties in identifying ingredients in these foods8.21 make many inadvisable for coeliac disease patients. Many such patients suffer long periods of poor health due to inadvertent consumption of gluten, even of special "gluten-free" foods or of pharmaceuticals (if they are based on poorly qualitycontrolled wheat starch).8.22 Despite the obvious health implications, many parents find it difficult to achieve compliance of their children (especiallly teenagers) with a gluten-free diet. Availability of a simple and inexpensive test kit for gluten in foods should potentially enable radical changes to the lifestyles and health of many coeliac disease patients. We thank the CSIRO Development Pool and Medical Innovations Limited, Labrador, Queensland, Australia, for financial support.

REFERENCES WT, Holmes GKT. Coeliac disease. Edinburgh: Churchill Livingstone, 1984. 2. Davidson AGF, Bridges MA. Coeliac disease: a critical review of aetiology and pathogenesis. Clin Chim Acta 1987; 163: 1-40. 3. Walker-Smith JA. The diagnosis of coeliac disease. Arch Dis Child 1979; 1. Cooke

54: 783. 4. Swinson CM, Levi AJ. Is coeliac disease underdiagnosed? Br Med J 1980; 281: 1258-60. 5. Homes GKT. Malignancy in coeliac disease—effects of a gluten-free diet. Gut 1989; 30: 333-38.

Jones A. Wheat sensitive—but not coeliac. Lancet 1978; ii: 1047. Campbell JA. Diet therapy of coeliac disease and dermatitis herpetiformis. World Rev Nutr Diet 1987; 51: 198-240. 8. Skerritt JH, Wrigley CW, Wilkinson W, La Brooy JT. Wheat starch and the gluten-free diet. Med J Aust 1987; 147: 262-63. 9. Ciclitira PJ, Ellis HJ, Fagg NLK. Evaluation of a gluten-free product containing wheat gliadin in products with coeliac disease. Br Med J 6. 7.

1984; 289: 83-84. 10.

Dissanayake AS, Truelove SC, Whitehead R. Lack of harmful effect of oats on small-intestinal mucosa in coeliac disease. Br Med J 1974; iv:

189-91. 11. Anand BS, Piris J, Truelove SC. The role of various cereals in coeliac disease. Quart J Med 1978; 47: 101-10. 12. Skerritt JH. Immunoassays of non-meat proteins in foods. In: Rittenburg J, ed. Development and application of immunoassay for food analysis. London: Elsevier, 1990: 88-125. 13. Skerritt JH, Smith RA. A sensitive monoclonal antibody-based test for gluten detection. Studies with cooked or processed foods. J Sci Food Agric 1985; 36: 980-85. 14. Skerritt JH, Hill AS. Monoclonal antibody sandwich enzymeimmunoassays for determination of gluten in foods. J Agr Food Chem 1990; 38: 1771-78. 15. Skerritt JH, Hill AS. Enzyme-immunoassay for the determination of gluten in foods: collaborative study. J Assoc Off Anal Chem (in press). 16. Lee JW. Preparation of gliadin by urea extraction. J Sci Food Agric 1968; 19: 153-56. 17. Meredith P. On the solubility of gliadin-like proteins. II. Solubility in aqueous acid media. Cereal Chem 1965; 42: 64-71. 18. Bos ES, Van der Doalan AA, Von Rooyn N, Schurrs AHWM, 3,3’,5,5’-tetramethylbenzidine as an AMES-test negative chromogen for horseradish peroxidase in enzyme-immunoassay. J Immunoassay 1981; 2: 187-204. 19. McNicholl B, Egan-Mitchell B, Fottrell PF. Variability of gluten intolerance in treated childhood coeliac disease. Gut 1979; 20: 126-32. 20. O’Donoghue DP, Swarbrick ET, Kumar PJ. Type I hypersensitivity reactions in celiac disease. Gastroenterology 1979; 76: 1211. 21. Bell L, Hoffer M, Hamilton JR. Recommendations for foods of questionable acceptance for patients with coeliac disease. J Canad Diet Assn 1981; 42: 143-58. 22. Challen RG, O’Shannassy RM. Gluten content of Australian pharmaceutical products. Med J Aust 1986; 146: 91-93.

Relation of central obesity and insulin resistance with high diabetes prevalence and cardiovascular risk in South Asians

The hypothesis that the high mortality from coronary heart disease (CHD) in South Asians settled overseas compared with other populations is due to metabolic disturbances related to insulin resistance was tested in a population survey of 3193 men and 561 women aged 40-69 years in London, UK. The sample was assembled from industrial workforces and general practitioners’ lists. In comparison with the European group, the South Asian group had a higher prevalence of diabetes (19% vs 4%), higher blood pressures, higher fasting and post-glucose insulin concentrations, higher plasma serum HDL and lower cholesterol triglyceride. concentrations. Mean waist-hip girth ratios and trunk skinfolds were higher in the South Asian than in the European group. Within each ethnic group waist-hip ratio was correlated with glucose intolerance, insulin, blood pressure, and triglyceride. These results confirm the existence of an insulin

resistance syndrome, prevalent in South Asian populations and associated with a pronounced tendency to central obesity in this group. Control of obesity and greater physical activity offer the best chances for prevention of diabetes and CHD in South Asian people. Introduction

Mortality and morbidity from coronary heart disease (CHD) are higher in people of South Asian (Indian, Pakistani, and Bangladeshi) descent settled overseas than in other groups.! In England and Wales during 1979-83, ADDRESS Department of Community Medicine, University College and Middlesex School of Medicine, London UK (P M McKeigue, MFPHM, B Shah, MB, Prof M. G. Marmot, FFPHM) * Present address Indian Council of Medical Research, Ansari Nagar, New Delhi 110029, India. Correspondence to Dr P. M McKeigue, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1 E 7HT, UK