GB2207921A - Hybridoma cells and monoclonal antibodies - Google Patents

Abstract

Hybridoma cell lines ATCC HB9798 produce monoclonal antibodies directed against omega gliadin protein of wheat and related proteins in rye and barley. Such monoclonal antibodies can be used in a number of improved methods for the detection and quantification of gluten in foods and other substances. The invention has important applications in monitoring the levels of cereal incorporated into processed meat and as an analytical method for deletion of cereal protein.

Description

HYBRIDOMA CELLS AND MONOCLONAL ANTIBODIES 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 ty using these monoclonal antibodies which are apecific for certain gliadin proteins.

As used in this apecification 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 sovent Not al @ of the hybridoma clones which result from fusing myeloma cells with antibody-producing cells are specific for the desired foreign substance or antigen (a substance with which the antibody teacts) since many nf the hybridomas will make antibodies which the inoculated animal has produced to react with other foreign substances. Also aiitibodies against th subject antigen will differ from clone to clone since antibodies produced by different cells may react with different antigenic determinants of the ssme molecule. Tn addition, wheat gliadin is a complex mixture of several dozen protein molectlles. Prom each clone, therefore, it is necessary to obtain the resulting antibody artd 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 i invention has very particular and important applications Firstly, cereal proteins are being increasingly utilised ill processed foods. In the meat products they are added as binders to improve texture and aid water and fat retention and act as extenders t lower the cost of the finishecl product.There are recommended maximum levels of cereal and other n@n-meat proteins for incorporation into processed meat (Codex Alimentarius Commission, UN Food and Agricuiture Organization/World Health Organization Standard tor 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 boon hated during processing.

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

while obvious souroes of these cereal proteins may be avoided, these proteins si:t often found in unexpected sources. In addition to use as a meat extender, wheat 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 other 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 related cereal seed storage proteins are insoluble in aqueous solution.Secondly, polyclonal antiser@ 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 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, parttcularly in the food processiny, quality control and clinical fields.

A preferred method of the present invention in which food antigens are detecied is particularly desirable sinee extensive sample preparation such as de-fatting, salt removal or freeze-drying should nol: be needed. The procedure is adaptable to reasonably large number of samples (several dozen), does not reguire expensive automated e.quipmenL and allows such assays to be performed by small as well as large laboratories. Futher, the present invention is suitable for cooked and processed as we: as raw foods and gliadin protein may (ve 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 detenlination 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 bnd 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 rois.tant of the flour proteins. The quality of heat resistance means that omega-gliadins are extractable from cooked food products whreas most proteins are denatured by a cooking process. The present antibodies -.y thus be used as an assay for gluten-type proteins in foodstuffs, even cooked foodstuffs such as processed msats and canned foods, as the proportion of the omega-gliadin proteins in.

gluten is fairly constant and determination of the omega-gliadin content of a foodstuff provides an accurate guide to the total gliadin or gluten content thereof.

Our Australian Patent 3B501/B5 describe a method of detection of gluten in foods by use of monoclonal antibodieti specific for omega and gamma-gliadins following the application of substances thought to contain 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 nver 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 prodt ivity (antibody concentration) in tissue culture and in ascites fluids in vivo. Such novel antibodies have omega-gliadin specificities and affinities sufficiently high for them to be effective in the quantitation of gluten in foods by means of an antigen-oompetition assay, or as the antigen-capture antibody in an antigen-capture-antibody-tag ("sandwich") enzyme-immunoassay format.

A particular feature of the diet for donor animals according to this invention is that it is glutenofree. Accordingly, in one aspect of this invention there is provided a hybridoma cell line derived by fusion of an antibody-producing cell from an animal maintained on a gluten free diet with a myeloma cell, said antibody having a particular specificity and affinity for omega gliadins.

Prior to isolation of the antibody-producing cells, the animal will usually be immunologically challenged with an antigen capable of generating an antibody against an omega-gliadin protein of wheat, which antibody does not react with other wheat gliadin proteins. The e antigen may be an omega-gliadin protein of wheat.

Preferably the diet is free of wheat, rye, barley and oats and consists of cracked maize, fish and meat meals, 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 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 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, cystein, glutathione, thioglycolate or similar.

Preferably (for quantitative determinaticn of gluten) the food extractant is 40% (V/V) ethanol in water. Preferably the extract is diluted in phosphate-buffered saline containing 0.05% Teeen 20 and 0.1% cold-water Teleostean fish skin gelatin.

A hybridara cell line according to this invention is preferably produced by the fusion of an antibody- producing 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 we characterised cell line of which a number are in common usage A hybridoma cell line consisting of. the above features, and antibodies according to t.he present invention are available at CSIRO Wheat Research Unit, worth Ryde, New South Wales, Australia and more particularly there is provided a hybridoma cell line having tulle American Type Culture Collection accession number ATCC corresponding to antibody clone 401/21..

In a further aspect of this invention there is 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 omega-gliadin protein is applied to a solid support to which is passively or covalently bound a icnown amount of purified gliadin antigen or omega gliadin specific monoclonal antibody. After washing, the product is detected by a convenient procedure such as enzyme assay, radioinimunoassay, fluorescence immunoassay, or chemiluminescence.

A preferred omega-gliadin-specific-monoclonal 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 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 gliadinrscated or monoclonal-antibody coated microwell plates, strips, tubes or other suitable solid support, a lyophilised standard or solution or preparation containing defined amount of gliadin protein, and reagents for product detection such as an enzyme-conjugated monoclonal antibody to gliadin, enzyme substrate, washing and colour development stopping solution, and non-specific binding blocking reagents. Alternatively, the product might be detected by e.g. agglutination, adherence, fluorescence, radioimmunoassay or chemiluminesoent prooedures.

The invention extends to a method for the detection of omega gliadin protein, armor a related protein from rye or barley, in a example by pseudo-siniultaneus sandwich EIISA, the method comprising contacting the sample, or an extract of the sample, with a blocking solution in contact with an antibody-coated solid support to form a first mixture, incubating the first mixture for a period of fran 1 to 10 minutes, mixing the first mixture, without intermediate washing, with an enzyme-conjugated monoclonal antibody specific for an omega gliadin, washing the solid support and exposing the solid support to a chromogenic substrate for the enzyme.

The wheats are part of a family of cereal grasses including the ryes, barleys, oats, maizes (oorn), 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 sane patients, possibly oats 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 rioe 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 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 ra:ige of yoods.

EXPERIMENTAL Cereal Samples Bread wheat (Triticum aestiyum, Timgalen cultiva@) was milled to produce flour. Crude gliadin was prepared from this flour by two extractions of the residue (from flour extractions with 10% (w/v) sodium chloride solution) with 10 volumes of 40% (w/v) ethanol at 200C (Onborne, The Proteins of the Wheat Kernel. Carnegie Inst.

Washington D.C. Publ. 84 (1907)). Homogeneous gelatin 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 chemical composition and is readily soluble in 40% (v/v) ethanol in water.

On. ega-gliadin immunogens were prepared by preparative 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) wer also prepared.

Bulh/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 q/kg soybean meal 50 g/kg fish meal 40 g/kg 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/kq Vitamin E 0.20 International Units/kg Vitamin B1 (thiamine) 0.325mg/kg Vitamin B2 (riboflavin) 28 ug/kg Vitamin B6 (pyridoxine) 28 ug/kg Vitamin B12 0.67 ug/kg Vitamin K 48 ug/kg Riotin 0.672 ug/kg Niacin 0.101 mg/kg Calcium pantothenate 0.123 mg/kg Folic acid 11.2 ug/kg Magnesium 2.32 mg/kg Iron 0.37 mg/kg Manganese 0.64 mg/kg Zin 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 those 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 duriiig 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 mou@@ in Freund's incomplete adjuvant two and four weeks later. Doses were divided with half given subeutaneously and half intraperitoneally. Three months later, three mice (with high serum anti-gliadin antibody) were given a booster intraporitoneal infection (0.5 mg yliadin) and the spleen; removed for fusion, four days later. Spleen cells were fused with an appropriate mouse myeloma cell line (SP2/0, Galfre et al.Nature 266 (1977) 550-552) using polyethyleneglycol. The details of the fusion protocol and culture techniques used have been described elsewhere (Shulman et al. Nature 276 (1978) 269-270). Fused cells were separated by limiting dilution and antibody-secretin: clones were subcloned by further dilution. For some sub@lones 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 the marker enzymes horseradish peroxidase or alkaline phosphatase.

Screening for antibodies of des Supernatants from culture wells after initial cloning were assessed for antibody activity by use of indirect enzyme linked immunosorbent assays sing antigen immobilized on polystyrene microwells as detailed (Skerritt et al. J. Cereal Sci. 2 (1984) 215-224; 8)cerritt and Martinuzzi, J. Immunol. Methods 88 (1986) 217-224; Skerritt and Underwood, Biochim. Biophys.

Acta. 674 (198G) 245-254). Monoclonal antibodies 401/21, 405j5, 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 t The preferred hybridoma cell line for performing this invention is 401/21.

Method Assay format 1 : Quantitative determinating of gluten 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 microqramjwell 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% (v/v) ethanol by 30 second homogenisation. If cocoa, chocolate, coffee or foods containing polyphenols are present, 5 - 10% Teleostean fish skin gelatin is added to the extractant.

and extracts held one hour at 20 C before dilution.

Extracts are diluted in PBS-0.05% Tween 20 - 0.18 Teleostean fish skin gelatin, either 50-fold, 500-fold or 2500-fold, depending upon the expected gluten content.

Gliadin standards are prepared by reconstituting to 0.5 mg/ml in 40% ethanol and dilution in PRS-Tween-gelatin to prepare standards of 5, 2.5, 1.25, 0.625, 0.313 and 0.156 micrograms per millilitre.

diluted fond extracts or gliadin standards are applied to wells of the microwell plate, incubated 30 minutes at 15-250C, washed, then omega-gliadin-specific monoclonal antibody labelled with marker enzyme such as horseradish peroxidase or alkaline phosphatase added and incubated 30 min at 15-250C. After washing, a chromegenic enzyme substrate is added. The gluten content is double the calculated gliadin content, as typically 50% 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 Absorbance Units above background) is 0.10 micrograms gluten per ml of incuhation mixture, which corresponds to 0.01% (w/w) gluten in the food sample when a 1 in 50 sample extract dilutton is used.When 1 in 5 sample dilution is used the detection limit iR increased t 0.001% (w/w) gluten (1 mg gluten per 100 g food). Gluten content of selected foo using 3 sandwich enzyme immunoassay with antibody 401/21 Typical results 1/(Dilution ELISA Gluten Coeliac Tested) Product % toxic@ Absorbance at 414 nm Gliadin response curve 5 ug/mL 1.394 2.5 1.051 1.25 0.756 0.625 0.485 0.313 0.302 0.156 0.172 Baked Goods Bread crumb 2500 0.831 7.3 +++ Sweet biscuit 2500 0.558 3.7 +++ Savoury biscuit 2500 0.543 3.5 +++ Crispbread 2500 0.279 1.5 +++ Corn flake crumb 2500 0.002 n.d. Baby foods No. 1 (beef.based) 50 0 n.d. No. 2 (chicken-based) 50 1.263 0.4 ++ Processed meats/smallgoods Cooked beef product 1 500 0.031 n.d. Cooked beef product 2 2500 0.916 10.0 +++ Ham and chicken sausage 500 0.354 0.4 ++ Salami 500 0.022 n.d. - Soups - vegetable No. 1 50 0.023 n.d. No. 2 50 1.269 0.4 ++ Confectionery Caramel candy 500 0.829 1.4 +++ Chocolate candy 500 0.177 0.2 + Pet food 1 500 0.015 n.d.

2. 500 0.331 0.3 Starches Brand name starch (0.24% total protein) 50 0.062 n.d. Generic starches 1 (0.32% total protein) 50 0.340 0.03 +/ Generic starches 2 (0.43% total protein) 50 0.740 0.12 + Other food proteins/protein Sources Lentils 50 0.002 n.d. Soy flour 50 00000 n.d. Egg 50 00000 n.d. Milk Powder 50 00000 n.d. Baking mixes labelled as "gluten free" Bread mix 1 50 0.080 n.d. 2 50 1.131 0.3 ++ 3 50 0.331 0.04 +/ Starch base (pre-gelatinized) 50 0.010 n.d. Breakfast cereals labelled as "gluten free" Rice-based 1 50 0.038 n.d. Muesli 2 50 0.626 0.09 + n. d.. not detectable Assay Format 2 : Quantitative determination of gluten by antigen-comoetition ELISA using monoclonal antibodies specific for omega-gliadins and related proteins in food The gliadin protein soluble in 40% ethanol is immobilized either passively or covalently at l ug/well to a solid phase consisting of flat-bottomed 96-well polystyrene microwell plates or strips.Following washing with ().05% Tween 20 in pllosphate-buffered saline, pH 7.2, microwells are stored in 1% bovine serum albumin in phosphate buffered saline (PBS) containing 0.01% sodbirrL, azide Food samples are extracted in 10 ml 40% (v/v) ethanol by 30 second homogenisation. If cocoa, chocolate, coffee or polyphenols are present, 5-10% Teleostean fish skin gelatin is added to the extractant and extracts held 1 hour at 200C before dilution.Extracts are diluted in PBS-0.05% Tween - 0.1% Teleostean fish skin gelatin, either 50-fold, 500-fold or 2500-fold, depending upon the expected gluten content. Gliadin standards are prepared by reconstituting to 0.5 mg/ml in 40% ethanol and dilution in PBS-Tween-gelatin to prepare standards of 5, 2.5, t.25, 0.625, 0.313 and 0.156 micrograms per millilitre.

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 added and incubated at 30 mien. at 15-250C.

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 result in an increased product absorbance while in assay format 2, decreases in product absorbance from values obtained in the absence of enalyte are obtained with increasing gluten analyte concentration.

The method can determine gluten in a wide rang 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 affect the test. Similarly no apparent phosphatase or phosphatase inhibiting activity was detect in foods.

The sensitivity of assay format 2, defined as Hie lowest concentration of antigen which gives an absorbance value 10% lower than the control performed in the abseenoe of antiqen is 2.7 ug/ml, corresponding to 0.054% gluten at a 1 in 10 sample diltition and 0.027% gluten at a 1 in 5 sample dilution.

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

Food samples are extracted by shaking chopped, ground or liquefied food well i a suitable gliadin extractant, including aqueous ursa, aqueous polar organic solvents such a dioxane, dimethylsulfoxide, ethylene glycol, or alcohols, acids, bass or detergents in some cases with the addition of A reducing agent. 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 chromogenic Substrate for the enzyme.Colour development indicates the presence of lOmg gluten or more per lOOg food.

However, by changing the antigen capture and enzyme labelled (tag) antibody and food sample extract concentrations or by chemical treatments to the solid phase or test sample, the sensitivity can be increased 10 fold to 1 mg gluten per lOOg food sample.

The best monoclonal antibody for these tests, both as a "capture" and "tag" (enzyme labelled antibody) is antibody 401/21. however other hybridomas secreting antibodies specific for omega-gliadin proteins in 40% (v/v) ethanol extracts of wheat grain and having appropriate affinity and titre will b suitable. In assay formats 1, 2 and 3 antibody reaction with grain protein from cereals toxic in coeliac disease, namely bred wheat (Triticum aestium), durum wheat (Triticum tu@gidum yar.

durum), rye (Secale cereale) and barley (Hordeum @ulgare) 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 for scope of the invention as broadly described.

Claims (17)

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 an omega-gliadin protein 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 urine species.

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

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

5. A hybridoma cell line according to claim 1 therein the animals female parent is fed a subatantially 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 5.

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 saniple thought to contain the protein to a support together with suitably labelled monoclonal antibody specific for cmega-glsadin 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 ot omega gliadins ill a food ample comprising : applying the food sample thought to contain the protein to a support; without or without applying appropriate washing steps, applying a suitably labelled monoclonal antibody specific for omega gliadin protein; applying appropriate washing steps; said support having hound, to it either passively or covalently a known amount of monoclonal antibody specific for omega-gliadin proteins; and detecting bound product by a convenient procedurtt.

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

10. A method according to claim 7 or 8 wherein the food sample is diluted in phosphate-buffered saline containing û.058 Tween 20 and 0.1$ cold water Teleostean fish skin gelatin.

1.1. A method according to claim 7 or 8 wherein the samples comprise cocoa, chocolate, coffee or polyphenol conta.ning foods, and wherein the samples are extracted in 40% (v/v) ethanol containing 5 to 108 cold water Teleostean fish skin gelatin.

12. A tnethod 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, ad after an incubation period of between 1 and 10 minutes are mixed 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 method 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 the immunoassay is lyophilized from a solution in 10 mM acetic acid for reconstitution in 40% (vlv) ethanol,

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

15. A kit for the quantitative detection of omega gliadin proteins and related proteins from rye or barley in foods or other substances comprising@- euitable solid support coated with gliadin protein or glladin 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 hybridoma cell line, or a monoclonal antibody food detecting omega-gliadin protein, substantially as hereinbefore described with reference to any one of i-he examples.

17. A metbod of producing a monoclonal antibody for detecting omega-gliadin protein substantially as he@einbefore described with reference to any one of the examples.