AU611921B2 - Gluten detection with monoclonal antibodies - Google Patents

Description

ODGED AT sUBOFFIC 8 JUL 1987 VelbourneJ jia P~ i COMMONWEALTH OF AUSTRALIA 4 r r l hit'' 1 Patent Act 1952 ,'rId ls coi rct for r

(ORIGINAL)

Class Int. Class Application Number :PI2985 Lodged :8 July 1987 Complete Specification Lodged Accepted Published rATENT m

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SYDI"izY Ct 0008? Collector of Public MOnlV' PATENT SUB OFICE

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Priority: Related Art Name of Applicant Address of Applicant Actual Inventor Address for Service COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION Limestone Avenue, Campbell, Australian Capital Territory, Commonwealth of Australia John Howard Skerritt and Amanada Susanne Hill F.B. RICE CO., Patent Attorneys, 28A Montague Street, BALMAIN. 2041.

Complete Specification for the invention entitled: "Improved monoclonal antibodies and test method for detection of gluten in foods" The following statement is a full description of this invention including the best method of performing it known to Us:- 2 The present invention relates to improved hybridoma cell lines which produce monoclonal antibodies known as 401/21, 405/5, 405/7, 304/13 and 413/13, and particularly to an improved method of detecting gluten in foods by using these monoclonal antibodies which are specific for certain gliadin proteins.

As used in this specification the term gliadin protein is applied to the group of proteins found in wheat gluten and which are soluble in a 30-85% ethanol in water 10 solvent.

Not all of the hybridoma clones which result from 0 fusing myeloma cells with antibody-producing cells are 00 specific for the desired foreign substance or antigen (a o a o° substance with which the antibody reacts) since many of .oo:o: 15 the hybridomas will make antibodies which the inoculated animal has produced to react with other foreign substances. Also antibodies against the subject antigen will differ from clone to clone since antibodies produced by different cells may react with different aiitigenic determinants of the same molecule. In addition, wheat gliadin is a complex mixture of several dozen protein molecules. From each clone, therefore, it is necessary to o obtain the resulting antibody and test its reactivity with the subject antigen mixture (gliadin) and to test its specificity by determining which particular gliadin proteins, if any, it recognizes.

The present invention has very particular and important applications. Firstly, cereal proteins are being increasingly utilised in processed foods. In the meat products they are added as binders to improve texture and aid water and fat retention and act as extenders to lower the cost of the finished product. There are recommended maximum levels of cereal and other non-meat proteins for incorporation into processed meat (Codex Alimentarius Commission, UN Food and Agriculture 3 Organization/World Health Organization Standard for Gluten-free foods (Stan 118-1981) and standards for processed meats Stan 88-1981, 89-1981, 96-1981, 97-1981, 98-1981, 117-1981) but it has been difficult to monitor such incorporation. The foreign cereal proteins must be distinguished from meat protein; this problem is complicated when the proteins have been heated during processing.

Secondly, a need for an analytical method for cereal 1o 0 protein arises because a significant number of individuals (between 1 in 300 and in 1 in 3000 Caucasians) cannot o tolerate certain cereals in their diets; of r cereal-sensitive conditions the best characterised is ool coeliac disease (Cook and Holmes, "Coeliac Disease", 15 Edinburgh: Churchill Livingstone, 1984) in which certain gluten proteins from wheat, rye and barley (and in a few of these patients, oats) damage the small-intestinal Soo lining resulting in malabsorption leading to severe nutritional deficiencies. The only means of ameliorating these symptoms is strict avoidance of these proteins.

While obvious sources of these cereal proteins may be avoided, these proteins are often found in unexpected sources. In addition to use as a meat extender, wheat 0 flour is often used as a thickening agent in soups and desserts. Barley proteins from malting may be found in beer and some alcoholic spirits as well as in some breakfast cereals. Gluten is often present in otheir products such as confectionery and as a tablet binder in analgesics.

The idea of using immunological methods to determine gluten in foods (Keiser and Mahler, Lancet 1 (1973) 67) is not new. However, earlier attempts have suffered from three major disadvantages. Firstly, immunochemical methods based on antibody and antigen diffusion in gels perform best with water-soluble proteins, yet gluten and 4 related cereal seed storage proteins are insoluble in aqueous solution. Secondly, polyclonal antisera and most monoclonal antibodies do not give reliable results after gluten proteins have been heated in baking or food processing due to the denaturation of these proteins.

Thirdly, differences in antibody levels and specificities between polyclonal antisera from individual animals make standardisation of those preparations for analytical purposes difficult. The application of the present 10 improved invention is in detecting the presence of gluten and has substantially none of these disadvantages and is therefore a substantial contribution to the art, o particularly in the food processing, quality control and clinical fields.

15 A preferred method of the present invention in which food antigens are detected is particularly desirable since extensive sample preparation such as de-fatting, salt oo removal or freeze-drying should not be needed. The 'o o procedure is adaptable to reasonably large number of samples (several dozen), does not require expensive So. automated equipment and allows such assays to be performed by small as well as large laboratories. Further, the present invention is suitable for cooked and processed as well as raw foods and gliadin protein may be extracted in a single step. That is, the method of the present invention relies on those gliadin proteins which are heat-stable and therefore not affected by such processes.

The method of the present invention is simple, inexpensive and reliable and allows either qualitative or quantitative determination of gluten in samples. Finally, the sensitivity of the test is readily altered by changing either antibody concentrations or food extraction volume.

The gliadins may be classified into four groups, alpha-, beta-, gamma- and omega-gliadins according to their mobility on cathodic starch or polyacrylamide electrophoresis gels. The antibodies according to this invention are preferably such that they will bind to certain of the omega-gliadins, but not to other wheat gliadins. The omega-gliadins are the electrophoretically least mobile of the gliadins. They are either free of sulfur or have a very low sulfur content and are the most heat resistant of the flour proteins. The quality of heat resistance means that omega-gliadins are extractable from cooked food products whereas most proteins are denatured #V 10 by a cooking process. The present antibodies may thus be o used as an assay for gluten-type proteins in foodstuffs, even cooked foodstuffs such as processed meats and canned Sa* foods, as the proportion of the omega-gliadin proteins in gluten is fairly constant and determination of the i ,15 omega-gliadin content of a foodstuff provides an accurate guide to the total gliadin or gluten content thereof.

Our Australian Patent 38501/85 describes a method of o detection of gluten in foods by use of monoclonal Voo, antibodies specific for omega- and gamma-gliadins 20 following the application of substances thought to contain e4 4S these proteins to a suitable support. While that application discloses the general concept of detection of gluten in foods using a monoclonal antibody specific to gamma- and omega-gliadin it only provides a method for detecting gliadin proteins comprising the application of samples thought to contain the protein to a suitable untreated protein-binding support.

The present invention offers an improvement over the prior art in that it has been discovered that by careful control of the diet of animals selected for donation of cells for hybridoma production, and the use of purified omega-gliadin antigens, it is possible to produce antibodies of vastly superior affinity and productivity (antibody concentration) in tissue culture and in ascites fluids in vivo. S~oh novel antibodies have omega-gliadin K p.

6 specificities and affinities sufficiently high for them to be effective in the quantitation of gluten in foods by means of an antigen-competition assay, or as the antigen-capture antibody in an antigen-capture-antibodytag ("sandwich") enzyme-immunoassay format.

A particular feature of the diet for donor animals according to this invention is that it is gluten-free.

Accordingly, in one aspect of this invention there is provided a hybridoma cell line derived by fusion of an 10 antibody-producing cell from an animal maintained on a a gluten free diet with a myeloma cell, said antibody having a particular specificity and affinity for omega-gliadins.

Preferably the diet is free of wheat, rye, barley and o oats and consists of cracked maize, fish and meat meals, o 15 cotton seed and soy bean meals, minerals, vitamins and trace elements.

Animals should be fed the modified diet during their lifetime until sacrifice, and preferably their female 0o parents should be fed the diet from mating until parturition.

-Preferably the antibodies according to this invention bind to certain of the omega-gliadin proteins of wheat but not to any of the other wheat proteins present in the food o o extract. Suitable food extractants include aqueous alcohols, urea, guanidine hydrochloride, dilute acids or alkalis, soaps or detergents, in some cases with the addition of a reducing agent including dithiothreitol, dithioerythritol, 2-mercaptoethanol, cysteine, I glutathione, thioglycolate or similar.

Preferably (for quantitative determination of gluten) the food extractant is 40% ethanol in water.

Preferably the extract is diluted in phosphate-buffered saline containing 0.05% Tween 20 and 0.1% cold-water Teleostean fish skin gelatin.

The hybridoma cell line according to this invention 7 is preferably produced by the fusion of an antibodyproducing cell and a myeloma cell derived from a murine species. The antibody producing cells are preferably spleen cells. Any suitable myeloma cell line may be used, however it is desirable to use a well-characterised cell line of which a number are in common usage.

The hybridoma cell line consisting of the above features, and the antibodies according to the present invention are available at CSIRO Wheat Research Unit, North Ryde, New South Wales, Australia and more particularly there is provided a hybridoma cell line deposited wi the American Type Culture Collection on June 29th 1988 and o assigned the ATCC designation HB 9798 corresponding to S antibody clone 401/21.

U° O 15 In a further aspect of this invention there is S"o provided a method for detecting gliadin proteins in foods and other substances. In this method a solution containing an appropriately diluted sample and a solution of a suitably labelled monoclonal antibody specific for omegagliadin protein is applied to a solid support to which is passively or covalently bound a known amount of purified o Vo gliadin antigen or omega gliadin specific monoclonal antibody. After washing, the product is detected by a convenient procedure such as enzyme assay, radioimmunoassay, .s 25 fluorescence immunoassay, or chemiluminescence.

A preferred omega-gliadin-specific-monoclonal o' antibody support or gliadin antigen support for use in the above method would take the form of polystyrene microwell plates, strips or test tubes. The support may however have other configurations and take the form of, for example tubes, elongate sticks or beads. Other materials for formation of the support include polyvinylchloride, nitrocellulose, nylon and glass fibre membranes. The antibody or antigen may be passively adsorbed onto the support or covalently bound, for example by prior .j/ 8 treatment of the support or antibody or antigen with a bifunctional organic reagent, transition metal ion-containing solution, or by exposure to acidic or alkaline conditions.

In a still further aspect of the invention there is provided a kit or kits for detecting gliadin proteins comprising a packet containing gliadin-coated or monoclonal-antibody coated microwell plates, strips, tubes or other suitable solid support, a lyophilised standard or 10 solution or preparation containing defined amount of Sgliadin protein, and reagents for product detection such 0 as an enzyme-conjugated monoclonal antibody to gliadin, °o enzyme substrate, washing and colour development stopping 0o a solution, and non-specific binding blocking reagents.

15 Alternatively, the product might be detected by e.g.

agglutination, adherence, fluorescence, radioimmunoassay or chemiluminescence procedures.

o o The wheats are parct of a family of cereal grasses 0* including the ryes, barleys, oats, maizes (corn), sorghums and rices. All of the seeds of these grasses contain prolamin proteins of which some of the wheat prolamins are known as gliadin proteins. Of these prolamins only those of wheat, rye, barley and in some patients, possibly oats o^ o are implicated in coeliac disease while those of rice and corn, for instance, are not so implicated. The antibodies of the present invention are, as has been pointed out above, specific for certain of the gliadins among the wheat proteins. They have, however, also been found to bind to certain of the prolamins of rye and barley but not to those of rice or corn. Thus the antibodies of the present invention may be used to test foodstuffs for the presence of flours from the grains implicated in coeliac disease.

Hereinafter given by way of example are preferred embodiments of the invention describing the preparation of

S.

9 the cell line and the antibody according to the present invention and the use of those antibodies to bind to gluten-like prolamins from a variety of grain species to determine the presence of these proteins in a range of goods.

EXPERIMENTAL

Cereal Samples Bread wheat (Triticum aestivum, Timgalen cultivar) was milled to produce flour. Crude gliadin was prepared from this flour by two extractions of the residue (from S flour extractions with 10% sodium chloride solution) with 10 volumes of 40% ethanol at 200C (Osborne, SThe Proteins of the Wheat Kernel. Carnegie Inst.

o° Washington D.C. Publ. 84 (1907)). Homogeneous gliadin 15 antigen was produced by dialysis of the pooled ethanol extract against 10mM acetic acid then freeze-drying. This antigen preparation is homogeneous in appearance and °o chemical composition and is readily soluble in 40% (v/v) Sethanol in water.

Omega-gliadin immunogens were prepared by preparative oo. polyacrylamide gradient gel electrophoresis using the method of Caldwell (Biochem. Physiol. Pflanzen. 179 (1984) 31-43). Other cultivars of bread wheat and of durum wheat (Triticum durum) were milled to produce wholemeal. Milled samples of rye (Seale cereale), barley (Hordeum vulgare) maize (Zea mays) and rice (Oryza sativa) were also prepared.

Expermental animal diet Blb/C male and female mice were maintained on a gluten.free diet consisting of: cracked maize 763.9 g/kg meat meal 70 g/kg cottonseed meal 55 g/kg soybean meal 50 g/kg fish meal 40 g/kg 1_I_~L_ limestone (crushed) 5 g/kg sodium chloride 5 g/kg dicalcium hydrogen phosphate 3 g/kg sunflower oil 2.5 g/kg Vitamin A 126 International Units/kg Vitamin D3 22.4 International Units/kg Vitamin E 0.20 International Units/kg Vitamin Bl (thiamine) 0.325 mg/kg Vitamin B2 (riboflavin) 28 ug/kg 10 Vitamin B6 (pyridoxine) 28 ug/kg Vitamin B12 0.67 ug/kg Vitamin K 48 ug/kg Biotin 0.672 ug/kg 0 4 Niacin 0.101 mg/kg 15 Calcium pantothenate 0.123 mg/kg Folic acid 11.2 ug/kg Magnesium 2.32 mg/kg o Iron 0.37 mg/kg o Manganese 0.64 mg/kg Zinc 0,35 mg/kg Copper 28 ug/kg Iodine 11,2 ug/kg Molybdenum 5,6 ug/kg Cobalt 0.2 ug/kg Ethoxyquin 560 ug/kg After three months these mice were mated, and after weaning their female progeny reared for one month on the said gluten-free diet. Such diet was used with these mice during the course of immunizations and until sacrifice.

Production of monoclonal antibodies Mice (BALB/C) were injected with purified-omega gliadin (from the wheat variety Timgalen). The initial immunization in 50% Freunds complete adjuvant (0.2 mg omega gliadin in 0.1 ml 16 mM acetic acid per mouse) was followed by 0.1 mg omega gliadin per mouse in Freund's i 11 incomplete adjuvant two and four weeks later. Doses were divided with half given subcutaneously and half intraperitoneally. Three months later, three mice (with high serum anti-gliadin antibody) were given a booster intraperitoneal injection (0.5 mg gliadin) and the spleens removed for fusion, four days later. Spleen cells were fused with an appropriate mouse myeloma cell line (SP2/O, Galfre et al. Nature 266 (1977) 550-552) using polyethyleneglycol. The details of the fusion protocol 0 10 and culture techniques used have been described elsewhere (Shulman et al. Nature 276 (1978) 269-270). Fused cells o o' were separated by limiting dilution and antibody-secreting clones were subcloned by further dilution. For some subclones of clone 401/21, hybridomas were grown as ascites tumours for 10 days in mice. Antibody from these ascites fluids was purified by ammonium sulfate precipitation then ion-exchange or hydroxylapatite adsorption chromatography, and in some cases labelled with ao° the marker enzymes horseradish peroxidase or alkaline phosphatase.

Screening for antibodies of desired specificity Supernatants from culture wells after initial cloning were assessed for antibody activity by use of indirect oo enzyme linked immunosorbent assays using antigen immobilized on polystyrene microwells as detailed (Skerritt et al. J. Cereal Sci. 2 (1984) 215-224; Skerritt and Martinuzzi, J. Immunol. Methods 88 (1986) 217-224; Skerritt and Underwood, Biochim. Biophys.

Acta. 874 (1986) 245-254). Monoclonal antibodies 401/21, 405/5, 405/7, 304/13 and 413/13 were of high titre (greater than 1/5000) and bound on immunoblots of gliadin proteins to discrete bands in the omega-gliadin region at the antibody concentrations used in the test for gluten in foods. The preferred hybridoma cell line for performing this invention is 401/21lWkcM st-eo. o t'lt kA" o1b 0 h 12 12 Method Assay format 1: Quantitative determinating <'.iten by capture-tag (sandwich) ELISA using monoclonal antibodies specific for omega gliadins and related proteins in food extracts The capture antibody 401/21 is immobilized either passively or covalently at 1 microgram/well to a solid phase consisting of flat-bottomed 96- well polystyrene microwell plates or strips. Following washing with 0.05% Tween 20 in phosphate buffered saline, pH 7.2, microwells are stored in 1% bovine serum albumin in phosphate buffered saline (PBS) containing 0.01% sodium azide.

Food samples are extracted in 10 ml 40% ethanol by 30 second homogenisation. If cocoa, chocolate, coffee 15 or foods containing polyphenols are present, 5 Teleostean fish skin gelatin is added to the extractant and extracts held one hour at 200C before dilution.

Extracts are diluted in PBS-0.05% Tween 20 0.1% o o Teleostean fish skin gelatin, either 50-fold, 500-fold or 2500-fold, depending upon the expected gluten content, °a Gliadin standards are prepared by reconstituting to mg/ml in 40% ethanol and dilution in PBS-Tween-gelatin to prepare standards of 5, 2.5, 1.25, 0.625, 0.313 and 0.156 micrograms per millilitre, Diluted food extracts or gliadin standards are applied to wells of the microwell plate, incubated minutes at 15-250C, washed, then omega-gliadin-specific monoclonal antibody labelled with marker enzyme such as Shorseradish peroxidase or alkaline phosphatase added and incubated 30 min at 15-25aC. After washing, a chromogenic enzyme substrate is added. The gluten content is double the calculated gliadin content, as typically of the protein content of wheat gluten is gliadin (Kent, "Technology of Cereals", Oxford Pergamon Press 1975).

The sensitivity of Assay format 1 (defined as 0.1 1. A hybridoma cell line produced by the fusion of an antibody producing cell from an animal maintained on a substantially gluten free diet with a myeloma cell, said hybridoma producing an antibody directed against /2 now 13 Absorbance Units above background) is 0.10 micrograms gluten per ml of incubation mixture, which corresponds to 0.01% gluten in the food sample when a 1 in sample extract dilution is used. When 1 in 5 sample dilution is used the detection limit is increased to 0,001% gluten (1 mg gluten per 100 g food).

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L; Li 42 tp 4-)N 44 0 4- 0 0 *H0 LOo0 0V4 044 Gluten content of selected foods detected using a sandwich enzyme immunoassay with antibody 401/21 Typical results l/(Dilution ELISA Gluten Coeliac Tested) Product %toxic? Absorbance at 414 rim Gliadin response curve 5 ug/mL 1.25 0.625 0 .313 0.156 Baked Goods Bread crumb Sweet biscuit Savoury biscuit Crispbread Corn flake crumb Baby foods No. 1 (beef-based) No. 2 (chicken-based) Processed meats/smaligoods Cooked beef product 1 Cooked beef product 2 Ham and chicken sausage Salami 2500 2500 2500 2500 2500 1.394 1.051 0.756 0.485 0.302 0.172 0.831 0.558 0.543 0 .279 0.002 0 1.263 0.031 0.916 0. 354 0.022.

7.3 3.7 n.d.

n.d.

0.4 n.d.

10.0 0.4 n~d.

500 2500 500 500 O 000 00 0 0*0 0 0 0 C CO 0 C 0 0 0 COO 0 0 C 0 00 0 0 0 0 0 0 0 0000 0 0 Soups -vegetable No. 1 50 0.023 n.d.

N.250 1.269 0.4 Confectionery Caramel candy 500 0.829 1.4..

Chocolate candy 500 0.177 0.2+ 0 Pet food 1500 0.015 n.d.

2. 500 0.331 0.3 0 Starches Brand name starch (0.24% total protein) 50 0.062 n.d.

L4 Generic starches 1 (0.32% total protein) 50 0..340 0.03 Generic starches 2 (0.43% total protein) 50 0.740 0.12 0 Other food proteins/protein sources -444 Lentils 50 0.002 n.d.

Soy flour 50 0.000 n.d.

04Egg 50 0.000 n.d.

S4-4 Milk Powder 50 0.000 n.d.

E0 Baking mixes labelled as "gluten free" Bread mix 1 50 0.080 n.d.

4J C 2 50 1.131 0.3 3 50 0.331 0.04 1 Starch base (pre-gelatinized) 50 0.010 n.d.

.PW Breakfast cereals labelled as "gluten free" (0 En 4J)Rice-based 1 50 0.038 n.d.

En.0 Muesli 2 50 0.626 0.09 n.d. .not detectable 0 U% 010u E-i

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16 Assay Format 2: Quantitative determination of gluten by antigen-competition ELISA using monoclonal antibodies specific for omega-gliadins and related proteins in food extracts The gliadin protein soluble in 40% ethanol is immobilized either passively or covalently at 1 ug/well to a solid phase consisting of flat-bottomed 96-well polystyrene microwell plates or strips. Following washing with 0.05% Tween 20 in phosphate-buffered saline, pH 7.2, 1 10 microwells are stored in 1% bovine serum albumin in phosphate buffered saline (PBS) containing 0.01% sodium S azide.

Food samples are extracted in 10 ml 40% ethanol o, a 0by 30 second homogenisation. If cocoa, chocolate, coffee i 15 or polyphenols are present, 5-10% Teleostean fish skin gelatin is added to the extractant and extracts held 1 hour at 20oC before dilution. Extracts are diluted in PBS-0.05% Tween 0.1% Teleostean fish skin gelatin, o'o either 50-fold, 500-fold or 2500-fold, depending upon the expected gluten content. Gliadin standards are prepared o by reconstituting to 0.5 mg/ml in 40% ethanol and dilution in PBS-Tween-gelatin to prepare standards of 5, 2.5, 1.25, 0.625, 0.313 and 0.156 micrograms per millilitre.

e. Diluted food extracts or gliadin standards are applied directly to wells; of the microtitre plate and omega-gliadin specific monoclonal antibody labelled with marker enzyme such as horseradish peroxidase or alkaline phosphatase -ded and incubated at 30 min. at 15-25oC.

After washing a chromogenic enzyme substrate is added.

Concentration-dependent product colour development is produced for the gliadin standard and extracts of foods containing gluten-like proteins from bread and durum wheat, rye and barley, while extracts of corn, rice and cereals not toxic in coeliac disease do not react. In assay format 1 increasing concentrations of gluten analyte

I

perform best with water-soluble proteins, yet gluten and 1 17 result in an increased product absorbance while in assay format 2, decreases in product absorbance from values obtained in the absence of analyte are obtained with increasing gluten analyte concentration.

The method can determine gluten in a wide range of baked and processed foods, meat products, beverages (including beers), pharmaceuticals and confectionery.

Corresponding assays to determine interference by enzymes have been performed, showing that while some foods contain high levels of endogenous peroxidase (such as certain vegetables, legumes and meats) this does not a affect the test. Similarly no apparent phosphatase or o o phosphatase inhibiting activity was detected in foods.

0 0 The sensitivity of assay format 2, defined as the 15 lowest concentration of antigen which gives an absorbance value 10% lower than the control performed in the absence of antigen is 2.7 ug/ml, corresponding to 0.054% gluten at a 1 in 10 sample dilution and 0.027% gluten at a 1 in o sample dilution.

Assay format 3: Rapid, semiquantitative "oa odetermination of gluten by pseudo-simultaneous sandwich ELISA using monoclonal antibodies specific for omega-gliadins and related proteins oo The capture antibody 401/21, specific for omega-gliadins and related proteins from rye and barley, is immobilized either passively or covalently to a solid-phase consisting of proteins, binding strips or dip-sticks or polystyrene test tubes. Following washing with 0.05% Tween 20 in phosphate-buffered saline, pH 7.2, the solid phase is stored in 1% bovine serum albumin (PBS) containing 0.01% sodium azide.

Food samples are extracted by shaking chopped, ground or liquefied food well in a suitable gliadin extractant, including aqueous urea, aqueous polar organic solvents such as dioxane, dimethylsulfoxide, ethylene glycol, or their mobility on cathodic starch or polyacrylamide

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-18 alcohols, acids, bases or detergents in some cases with the addition o a reducing agent. A drop of the food extract is added directly to the solid phase, contents mixed 1 to 10 minutes then a drop of enzyme-labelled monoclonal antibody specific for omega-gliadins is added and contents mixed for another 1 to 5 minutes. The tube or other solid phase is emptied and washed well using tap water. The solid phase is then exposed to a specific o chromogenic substrate for the enzyme. Colour development 0 indicates the presence of 10mg gluten or more per 100g Sfood.

o0 However, by changing the antigen capture and enzyme 0 00 labelled (tag) antibody and food sample extract o concentrations or by chemical treatments to the solid 0 0 phase or test sample, the sensitivity can be increased fold to 1 mg gluten per 100g food sample.

0o aO The best monoclonal antibody for these tests, both as °o°o a "capture" and "tag" (enzyme labelled antibody) is antibody 401/21. However other hybridomas secreting 20 antibodies specific for omega-gliadin proteins in ethanol extracts of wheat grain and having appropriate affinity and titre will be suitable. In assay 0 0 I o 0 formats 1, 2 and 3 antibody reaction with grain proteins from cereals toxic in coeliac disease, namely bread wheat (Triticum aestium), durum wheat (Triticum turgidum var.

durum), rye (Secale cereale) and barley (Hordeum vulgare) were observed. No reaction with maize (Zea mays) and rice (Oryza sativa) grain proteins were noted.

The method of the present invention can be directly applied to kits for the detection of said gliadin proteins.

It will be recognised by persons skilled in the art that numerous variations and modifications may be made to the invention as described above without departing from the spirit or scope of the invention as broadly described.

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Claims (19)

1. A hybridoma cell line produced by the fusion of an antibody producing cell from an animal maintained on a substantially gluten free diet with a myeloma cell, said hybridoma producing an antibody directed against omega-gliadin proteins of wheat, which antibody does not react to other wheat gliadin proteins.

2. A hybridoma cell line according to claim 1 wherein the animal is of a murine species.

3. A hybridoma cell line according to claims 1 or 2 wherein the antibody producing cell is derived from a spleen.

4. A hybridoma cell line as claimed in claim 1 wherein the hybridoma cell line is No. 401/21 (ATCC No.

HB qtq) A hybridoma cell line according to claim 1 wherein the animal's female parent is fed a substantially gluten free diet from the time of mating until parturition.

6. A monoclonal antibody specific for omega-gliadin protein and related proteins from rye or barley produced from a hybridoma cell line as claimed in any one of claims 1 to

7. A method for the quantification and/or detection of omega gliadin proteins and related proteins from rye or barley particularly in a food sample comprising: applying the food sample thought to contain the protein to a support together with suitably labelled monoclonal antibody specific for omega-gliadin protein; with or without applying appropriate washing steps; said support having bound to it either passively or covalently a known amount of purified gliadin antigen; and detecting bound product by a convenient procedure.

8. A method for the quantification and/or detection of omega gliadins in a food sample comprising: applying the food sample thought to contain the protein to a support; without or without applying 4J 4 n~ ar[IUIuy Uor adiigeu may Ue pIcIiUJLveU-Ly cau ueu(U UIIL- Lilt support or covalently bound, for example by prior bound to it either passively or covalently a known amount 20 appropriate washing steps, applying a suitably labelled monoclonal antibody specific for omega gliadin protein; applying appropriate washing steps; said support having bound to it either passively or covalently a known amount of monoclonal antibody specific for omega-gliadin proteins; and detecting bound product by a convenient procedure.

9. A method according to claim 7 or 8 wherein the "o procedure for detecting bound product is selected from the O group consisting of: o 0 enzyme immunoassay, radio immunoassay, fluorescent 0 immunoassay, chemiluminescence, agglutination and C° o combinations thereof. oo

10. A method according to claim 7 or 8 wherein the food sample is diluted in phosphate-buffered saline containing 0.05% Tween 20 and 0.1% cold water Teleostean fish skin 0 gelatin. i'

11. A method according to claim 7 or 8 wherein the samples comprise cocoa, chocolate, coffee or polyphenol containing foods, and wherein the samples are extracted in ethanol containing 5 to 10% cold water Teleostean fish skin gelatin.

12. A method for the rapid detection of omega-gliadin protein and related proteins from rye or barley in foods and other substances by pseudo-simultaneous sandwich ELISA wherein, undiluted food extract is added to an antibody coated solid support containing blocking solution, and after an incubation period of between 1 and 10 minutes are Smixed together, without intermediate washing steps, with an enzyme conjugated monoclonal antibody specific for omega-gliadins, said solid support is washed and exposed to a specific chromogenic substrate for the enzyme.

13. A mAthod for the quantitative detection of omega gliadin protein as claimed in any one of claims 7, 8 or 12 wherein a homogeneous gliadin antigen standard for use in embodiments of the invention describing the preparation of 21 the immunoassay is lyophilized from a solution in 10 mM acetic acid for reconstitution in 40% ethanol.

14. A kit for the quantitation and detection of omega gliadin protein and related proteins from rye and barley in foods or other substances comprising: a standard solution or preparation containing a defined amount of gliadin protein and a monoclonal antibody directed against omega gliadin protein.

A kit for the quantitative detection of omega gliadin proteins and related proteins from rye or barley in foods Sor other substances comprising:- a suitable solid support coated with gliadin protein or gliadin monoclonal antibody, a standard solution or preparation containing a defined amount of gliadin protein, labelled monoclonal antibody directed against omega gliadin proteins, and where the label is an enzyme the kit further includes a substrate specific for that enzyme.

16. A method according to any one of claims 7, 8, 12 or 13 wherein the monoclonal antibody is according to claim 6.

17. A kit according to any one of claims 14 or 15 wherein the monoclonal antibody is according to claim 6.

18. A hybridoma cell line, or a monoclonal antibody for detecting omega-gliadin protein substantially as hereinbefore described with reference to any one of the examples.

19. A method of producing a monoclonal antibody for detecting omega-gliadin protein substantially as hereinbefore described with reference to any one of the examples. DATED this 12th day of December 1990. COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION Patent Attorneys for the Applicant: 4 F.B. RICE CO. Li