J . Sci. FoodAgric. 1985,36,987-994

A Sensitive Monoclonal-antibody-based Test for Gluten Detection: Quantitative Immunoassay

John H. Skerritt

CSIRO Wheat Research Unit, North Ryde, NSW2113, Australia

(Manuscript received 13 February 1985)

An enzyme-coupled monoclonal antibody has been used to quantify ‘gliadin-like immunoreactivity’ in a variety of foods. Small discs of nitrocellulose are soaked in food extract or a series of standard gliadin solutions, and incubated with antibody and an enzyme substrate yielding a soluble product. By use of a photometer, standard curves for gliadin may be constructed and the apparent gliadin content of samples calculated. The reproducibility and reliability of the procedure were examined using a variety of common foods and food proteins. The limit of detection for wheat gliadin was approximately 20,ugiml extract; gliadin levels in excess of this value were found in some ‘gluten-free bread mixes’. and starch sources. The overall time for analysis is 5-6h, although for large numbers of samples, overnight blocking of non-specific antibody binding may be used. It is possible that a ‘library’ of enzyme-linked monoclonal antibodies could be de- veloped as useful tools for specific food analysis.

Keywords: Monoclonal antibodies; gluten detection; immunoassay; coeliac disease.

1. Introduction The previous papers’,’ describe the development of an enzyme-conjugated antibody-based assay for gluten in foods, and semi-quantitative estimates of gluten content were made by analysis of the staining of nitrocellulose strips upon which dilutions of food extracts had been immobilised. This semiquantitative ‘spot test’ is adequate in many instances where a rough guide to gluten content is needed, but exact quantification may be required in other cases such as regulation of cereal content of processed meat and determination of gluten in trace quantities, e.g. in starches. Using an enzyme substrate yielding a soluble product, it is possible to develop this test for quantitative determination of gluten (as ‘gliadin-like immunoreactivity’) in foods. An enzyme- immunoassay using 96-well plastic plates and conventional antisera to soya proteins has been developed for their quantification in soya isolates, concentrates, extrudates and soya-based meat substitute^,^ but the test has not been applied to commercial foodstuffs. In preliminary experiments, gliadin binding to the plastic plates commonly used in quantitative immunoassays was found to be low and strongly affected by solvents. An alternate solid phase, nitrocellulose, showed good binding of cereal proteins extracted from foods using urea-based solutions.‘,* Therefore, a quantitative test for gluten in foods was developed, based on immobilisation on food proteins upon nitrocellulose. Such a test would allow quantitation of gluten when present in trace amounts and could thus have applications in quality control and food monitoring.

2. Methods 2.1. Cereal and food samples Extracts of cereals and foods, using six volumes of 2 M urea containing 0.2% dithiothreitol (D’IT) were prepared as earlier described.’V2 Sets of gliadin standards containing iO000, 3000, 1000,300, 100, 30, 10, 3 and Opg ml-’ in this solvent were prepared for each experiment.

987

988 John H. Skerritt

2.2. Antigen immobilisation and enzyme-immunoassay Nitrocellulose discs (Schliecher and Schull, 0.45pm pore size, 6 mm in diameter), prepared using an office punch, were soaked for 1 h in the supernatant from centrifuging (15000xg, 10min) of the food or cereal extract. Coated discs were then placed individually in 3ml plastic tubes, treated with 200pl of 1% KOH for 5min at room temperature and washed once with phosphate-buffered saline (PBS, 2 ml). Non-specific antibody binding was blocked by overnight incubation at 37°C in 200pl of 3% bovine serum albumin (BSA) in PBS. Following removal of blocking solution, 75pl anti-gliadin monoclonal antibody (clone 22/244-horseradish peroxidase (HRP) conjugate diluted 1 : l O O with 3% BSA in PBS) was added to each tube and incubated for 90min at 37°C. Discs were then washed (3xlOmin) with PBS containing 0.05% BSA and 0.05% Tween 20, dried, and horseradish-peroxidase substrate added. The most suitable substrate was found to be ABTS (2,2’-Azino-di-[3-ethylbenzthiazoline sulphonate (6)] diammonium salt), 1 mg ml-’ in 0 . 1 ~ sodium citrate-citric acid buffer, pH 4.2, containing 0.006% H202. One millitre of substrate solution was incubated per disc at room temperature for 15min then colour development was stopped by addition of 1 ml 20 mM sodium azide in 0. IM citric acid. Absorbance was read at 410nm.

2.3. Heat treatment of gliadin or flour Either gliadin (1 mg/200pI) or flour (1 mg/2pl) was suspended in water and heated in sealed tubes in a boiling water bath for various periods of time. Following cooling, extractant was added to a final concentration of 2~ urea-0.2% DTT and a final volume of 2ml mg-’ gliadin or 6pl mg-’ flour and extracted for 3 h at room temperature. Gliadin samples were assayed undiluted, while flour samples were diluted twenty-fold in extractant before assay. Apparent gliadin-like immunoreactivity at each time point was calculated from a standard curve for gliadin as described below.

2.4. Test baking Miniature loaves containing 35 g Timgalen flour, 0.60g sodium chloride, 0.17g improver (Dobrim 30/100) and 0.84g yeast were baked as described e l~ewhere .~ Loaves were dried for seven days at room temperature, and crumb and crust, separated from each loaf, were ground. Powders and samples of fldur were extracted for 3 h in 2M urea4.2% DTT and extracts were diluted fifty-fold in the same solution. The gliadin content of these samples was calculated from the standard curve. Results were corrected for the moisture content of each sample, determined by exhaustive freeze-drying.

2.5. Calculation of gliadin-like immunoreactivity In all experiments, food and cereal samples and gliadin standards were assayed in quadruplicate with antibody (tubes (A)). Appropriate ‘blank’ tubes in duplicate were run as follows, and incubated with substrate solution: (B): food sample (antigen) but no antibody, (C): no antigen but including antibody, and (D): no antigen and no antibody.

The absorbance difference due to endogenous peroxidase activity is therefore equal to (B) minus (D), while the difference due to non-specific binding of enzyme-labelled antibody to tubes and discs is represented by (C) minus (D). The colour due to antibody binding to antigen plus endogenous peroxidase activity is equal to (A) minus (C). Therefore, colour development due to antibody-antigen binding alone is equal to [(A) minus (C)] minus [(B) minus (D)]. In each assay a standard curve for gliadin was constructed and the concentration of gliadin-like immunoreactiv- ity was calculated for each test sample replicate by reading corrected absorbance values from this curve.

3. Results 3.1. Choice of enzyme substrate Preliminary experiments demonstrated the suitability of ABTS as a substrate for the HRP-

Antibody-based test for gluten 989

antibody conjugate. Another common HRP substrate, o-phenylenediamine, yielded unaccept- ably high backgrounds, possibly arising from spontaneous oxidation upon contact with the tube and disc surfaces. A 15min incubation time at room temperature appeared quite sufficient; colour development was linear with time over this period and continued for at least 1 h if a stopping solution were not added. There was no significant change in absorbance of tubes stored in the dark at 4°C for 24 h after addition of stopping solution.

3.2. Sensitivity and reproducibility of the assay In each experiment, a standard curve for gliadin was constructed. Figure 1 shows four standard curves for successive experiments conducted in different weeks; the shapes of the curves are quite similar. While 3 and lOpg ml-' gliadin did not yield absorbance differences, significant ( R 0 . 0 5 , Student's t-test on replicates) absorbances were seen at 30pg ml-' in five of six experiments. Absorbance plateaus occurred at gliadin concentrations between 3000 and 10 OOOpg ml-'.

Figure 1. Quantification of gliadin by enzyme immunoassay. The results of separate assays are indicated using different symbols. Error bars on quadruplicate determinations are omitted for clarity on Figures 1 , 2 and 3; typically standard errors were approximately 5% of the mean.

7 0.60

0.40

2 0.20 a

0 L

Similarly shaped curves were seen in three experiments using increasing concentrations of a bread-wheat flour extract (Figure 2). The absolute absorbance values obtained with known dilutions of flour extract varied slightly, with different batches of reagents over a two month period. It is important, therefore to set up standard curves for gliadin in each assay. The procedure could detect gliadin in flour down to 11333 dilution of the standard extract, and produced increasing absorbance with increasing proportions of flour extract until a 1/10 dilution was reached. On the basis of these experiments, extracts of gluten-rich food samples such as wheat cookies were routinely diluted 1/50 before analysis.

Comparison of urea-DTT extracts of various cereals (Figure 3) revealed that the method had similar sensitivity to bread and durum wheat, while the threshold for rye or barley meal detection was between 1:330 and 1:lOO. Much lower absorbance values were found with oats, rice and maize. Undiluted oat-protein extract did, however, yield double the absorbance value of maize

:: 2 0.20

Figure 2. Reproducibility of detection of gliadin in a flour extract. The results of separate assays are indicated using different symbols.

0.03 0.1 0.3 1.0 3.0 10 30 100,

- E ~

c

Figure 2. Reproducibility of detection of gliadin in a flour extract. results of separate assays are indicated using different symbols.

The

Flour extract (% initial)

65

990 John H. Skerritt

0

40 t I? A’.

00301 0 3 1 0 3 0 10 30 100

Figure 3. Assessment of ‘gliadin-like immunoreactivity’ in various cereals. Cereal extracts are indicated as follows: bread wheat, 0 durum wheat, A rye, A barley, oats, 0 maize 7 rice. The cereals were all assayed together.

Cereal extract (%initial)

or rice. In one experiment, lpl volumes of the cereal extracts and their dilutions were applied to discs instead of soaking the discs in the extracting solution. The assay was less sensitive, the limit of detection being 100-300ng gliadin. For each cereal studied, lpl of an undiluted extract produced a greater absorbance value than lpl of extract diluted 1 in 3.3 or 1 in 10. This approach would be suitable for comparison of samples known to be higher in gluten.

3.3 Effects of heating or baking upon gluten detection Vastly different levels of gliadin immunoreactivity were found in urea-DTT extracts following heating of water-gliadin or of water-flour suspensions. In the case of flour, 95% of immunoreac- tivity was lost from the extract following 10min heating (Figure 4), while the immunoreactivity of gliadin heated in the same way was unaltered. There was little further change after heating for longer periods. The effects of baking upon the sensitivity of gluten detection were studied by the preparation of three experimental loaves. Following correction for non-flour components, equivalent weights of the dried crumb portion contained 62+8% (meanks.e.m.) and the crust portion 89k 10% of the gluten-like immunoreactivity of the original flour samples.

3.4. Endogenous peroxidase activity of foods In the absence of added antibody, the absorbance difference between discs soaked in food extracts and those soaked in extractant only is presumably due to oxidation of added substrate by food enzymes. Only one food, soya beans (from two sources) produced significant colour in the absence of enzyme labelled antibody. A soya ‘flour’ extract yielded a mean 410nm absorbance value (A410) of 0.179k0.022 (three quadruplicate determinations) while milled soyabeans yielded a mean A410=0.452f0.012 ( n = 3 ) . This activity was abolished upon heating the preparations for 10min at 100°C.

All other foods had corresponding A410 values between -0.020 and +0.020. By comparison, 20pglml gliadin, taken as the threshold of detection, typically produced A4,0>0.040. This threshold was set since the standard deviation of the mean of absorbance values from discs containing no gliadin in each experiment was less than one-half of this value.

- - E 100

Figure 4. Effects of heating at 100°C on extractable immunoreactivity in: flour and gliadin.

(3 0 1020 50 I00 Time (min)

Antibody-based test for gluten 991

3.5 Gliadin-like immunoreactivity in various foods A variety of common foods was examined for apparent gluten content (Table 1). Each food was assayed several times so that the reproducibility of the assay could be assessed. Trace amounts of immunoreactivity were found in one of two starch preparations and one of two speciality gluten-free bread mixes examined. A variety of common food components (milk, egg, soya) did not show specific antibody binding. A commercial malt preparation, which presumably contained residual peptides from barley hordein, yielded a positive result. Raw meats produced a weak positive reaction; this effect was found for several samples of lamb, beef and pork. It is possible

Table 1. Quantitation of gliadin-like immunoreactivity in foods

Food

Powders Wheat-starch I Wheat-starch I1 Gluten-free bread mix I

Gluten-free bread mix I1

Milk powder Egg powder Malt Icing sugar Soya flour Soya beans

Meats Lamb (raw) Beef (raw) Beef (processed) Pork (raw) Processed porW lamb/cereal

Processed beef/ cereal

Sausage

soups I (vegetable) I1 (beef) I11 (beef/

IV (chickenkorn) vegetable)

Baked goods Bread crumb Bread crust Rice bread Wheat cookie Wheatirye cookie Rice cookies

Gluten presence

tr tr tr

tr

N N tr N N N

N N N N Y

Y,

Y

N N Y

tr

Y Y tr Y Y N

Corn breakfast cereal tr Rice breakfast cereal N

Individual assay results (ug ml-' extract) Mean

-

0

0 33f11

30 f5

0 0

0 0 0

144f10

30f7 47f 7

59f14 117f34 147f7 160f38 221f68 74 f9

169 233

0

0 0

73f18

0

3120f800 4400f860

36f15 1100+200 2750f200

87f19 0

0

0

0 57f19

35f5

0 0

0 0 0

75f23

35f11 49 f 10

42+10 540 f 200

530f140 260+53 73f22

179f35

0

0 0

181f15

0

4 140f 740 8260f 1380

62f18 1010f370 1890k 180

25f15 84f21 0

0

0 30f12

48t10

0 0

0 0 0

35f12

47 f7 38f25

28f6 403 f 28

783f76

232f33

0

0 0

135f44

0

2900f600 2960 f 540

50f10

0

0 126f45

0 40 0

38

0 0

85 0 0 0

37 45 0

43 302

39 1

145

0 0

130

0

3390 5210

49 1055 2320 120

99 0

Gliadin

food mg g-'

0 0.24 0

0.23

0 0 0.50 0 0 0

0.22" 0.2T 0 0.26" 1.81

2.35

0.87

0 0 0.78

0

20.3 31.3 0.29 6.3

13.9 <0.12

0.59 0

Food compositions checked with manufacturer. Presence of gluten indicated Y =yes, N=no, tr=trace A result of zero indicates less than 20pglml gliadin-like immunoreactivity. Foods were extracted as is

"Gliadin-like immunoreactivity lost after 10 min boiling. (not dried).

992 John H. Skerritt

that this effect is due to the presence of gluten fragments in the sera of these animals; such fragments, presumably of dietary origin, have been found in human sera.6 This is unlikely to be a problem in examination of processed meats for gluten since heating of these meats for 10min abolished all immunoreactivity .

Gluten could readily be detected in three commercial samples of processed meat and cereal, with a canned beefkereal preparation having a mean assay of 2.35 mg gliadin per gram food. On the basis of flour being about 4% gliadin, this would be equal to about 6% flour in the product. Due to gelatinisation upon heating it is likely that the actual value in this case is somewhat higher. Separate 3 g samples were cut from different parts of a 25G500 g piece of processed meat for each assay. The significant differences in assayed gliadin content between these slices may reflect an uneven distribution of flour in the finished product. The within-assay error for replicates was much lower, being about 20% of the mean for food samples and 5 1 0 % for cereal samples.

As expected, high levels of immunoreactivity were found in a number of baked goods; while a rice breakfast cereal was negative a maize cereal contained a small level of gliadin-like reactivity, presumably due to added malt.

4. Discussion

The method of soaking discs in aliquots of cereal and food extracts is extremely simple yet produces results with reasonable reproducibility. The 'disc-in-tube' method was preferred over enzyme immunoassay microtitre plates tor quantitative gluten assay, although the latter is slightly more amenable to automation. Although cereal proteins do adhere to plastic microtitre plates and such plates are suitable for screening for anti-prolamin antibodies in humans and experimental the nature of prolamin interactions with polyvinyl carbonate plastic and with nitrocellulose were found to differ. Using the same monoclonal antibody, the relative colour intensities produced by extracts (from equal masses of cereal meals) on plastic plates were as follows: Rye+bread wheat>barley =oats=durum wheat>rice=maize. The limit of detection for rye was also at least thirty-fold lower than bread wheat, while detection of durum wheat was only three-fold more sensitive than rice or maize. This cereal spectrum is quite different from that encountered for coeliac sensitivity or most other cereal intolerance conditions. In addition, since prolamin binding to plastic is very dependent on the solvent used' the presence of fats, salts or other food proteins in crude food extracts may have significant effects on antigen binding.

The nitrocellulose-disc method had more than adequate sensitivity for detection of 'coeliac- toxic' levels of flour protein. The toxic level set down by Australian convention," (i.e. less than 0.3% protein from wheat, rye, barley and oats in a food) is equal to about one part in 40 of flour. The assay is able to detect levels considerably below this for bread and durum wheats, barley and rye but not oats. The toxicity of the latter cereal in coeliac condition is in some doubt;"." indeed, dietary substitution of wheat by oats is often practised.

In the case of wheat protein, 0.3% protein in a food would correspond to a gliadin content of about 1.2mg g-I. Apart from the wheat-based baked goods, the processed meat products and one soup contained near (or in excess of) this level of gliadin immunoreactivity. This level is arbitrary and it is known that many coeliacs are sensitive to lower levels of g1~ ten . I~ The 'plateau' on the gliadin or flour concentrationiabsorbance curve is presumably due to antigen saturation of the nitrocellulose discs. While the maximal binding capacity of nitrocellulose for gliadin has not been accurately assessed, for serum albumin it has been estimated at about 80pg cm2.I4

Both the absolute and relative sensitivity of the assay for the various cereals are similar to those observed for the horseradish-peroxidase conjugated antibody in the semiquantitative assay, using 4-chloro-1-naphthol as the enzyme substrate.* As for that assay, the spectropho- tometric assay sensitivity could be altered if desired by increasing or decreasing food extraction volumes and antibody concentrations. A recommended procedure for quantitation of gluten is given in Table 2.

Antibody-based test for gluten 993

Table 2. Recommended procedure for gluten determination-quantitative method

1. Extract food sample with 6 vol 2~ urea-O.2% DTT, for 3 h at room temperature. Prepare gliadin standards simultaneously. Centrifuge and make appropriate dilutions of supernatants.

2. Soak nitrocellulose discs (6mm diameter) in food extracts, dry and transfer to test tubes. 3. Treat discs with alkali, wash then block free protein-binding sites with serum albumin (1 h at 45°C or overnight at 37°C). 4. Incubate discs (90min) with antibody-peroxidase conjugate. 5. Wash discs (3x5 or lOmin), add enzyme substrate (ABTSH,O,). After 15 min add stopping reagent and measure colour

intensity. 6. Gluten content of samples calculated from the standard curve for gliadin.

It must be stressed that while the assay should detect coeliac-toxic cereal species in foods, the assay relies on the detection of the heat-insensitive prolamins rather than those most toxic to coeliac patients. In the case of wheat, there is evidence that higher mobility prolamins (i.e. alpha- and beta-gliadins) have greater but it is unlikely that any gliadin fractions are devoid of toxicity. ”J*

A radioimmunoassay for alpha- and beta-gliadins has been developed,” and while this assay has been used to determine gluten in flours and in some ‘gluten-free’ breads,20 the greater heat lability of these proteins would lead to gross underestimation of gluten content in most processed foods. In addition, this radioimmunoassay did not detect barley and rye, cereals with appreciable coeliac toxicity. Other workers21,22 have optimised separate ELISA assays for alpha-gliadin and whole gliadin, but these assays have the same disadvantages; as little as 0.5% of the original gliadin was detectable after baking in some cases.

While it is possible to express analytical results in terms of ‘gliadin-like immunoreactivity’ by interpolation of individual values on a standard curve for gliadin prepared from a common variety of bread wheat, the gliadin or gluten contents are not being assessed on an absolute basis. There are slight differences in the degree of reactivity of the monoclonal antibody with different wheat cultivars but the antibody did bind to some low mobility prolamins in every bread, biscuit or spaghetti wheat cultivar examined.

The development of hybridoma technology has led to the use of monoclonal antibodies for clinical use in detection of drugs and hormones in serum,23 and in agriculture the development of methods for determination of viruses in crops.24 However, the present studies are the first application of a monoclonal antibody to the detection of species in foodstuffs. Enzyme irnrnunoassays (presently using conventional antisera) are useful in sensitive assays for detection of foreign species in meat products2’ and bacterial and mycotoxin contamination.26327 It is expected that the specificity and reproducibility of hybridoma antibodies as reagents will allow their use in these and many other applications in food science.

Acknowledgements The author is grateful to Dr C.W. Wrigley for helpful advice and to the Wheat Industry Research Council of Australia for financial support.

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