Field of the Invention The present invention relates to methods for identifying allergens in genetically modified organisms, and to animal models and compositions useful in practicing the method.

Background Of The Invention Genetic modification through biotechnology allows an organism to produce a particular trait, i. e., express a protein or other characteristic, which is novel for that organism. A particular issue raised by critics and other observers of the debate about genetically modified foods relates to new proteins produced in genetically engineered foods, with the expressed concern that these could act as allergens, either by themselves or in unintended ways in humans. In the case of a heterologous protein, allergenicity may be due to processing of proteins (e. g., glycosylation by the transgenic organism), or may be due to native proteins in the transgenic environment.

This concern derives from the fact that genetic engineering is different from traditional breeding, in that genetic engineering can move genetic material between completely unrelated plant species, and even between the plant, animal and microbe kingdoms in ways that could never occur in nature. Thus, even for proteins not known to be allergenic, there are concerns that when expressed in a genetic construct in a new organism, the proteins may interact with existing proteins in unexpected ways, or otherwise alter the metabolism of the food producing organism, causing it to produce new allergens or toxins.

The food-safety issues raised by the application of gene technology to some foods have yet to be satisfactorily addressed. In some jurisdictions this has prompted calls to introduce new food labeling requirements, to help consumers identify genetically modified foods. In one recent event the sale of a gene-altered corn seed variety was halted by the owner of the variety, over concerns about human safety when the corn, not approved for human consumption, inadvertently was introduced to the human food chain (Barboza, 2000). The references cited herein are listed after the Examples and before the claims.

In a May 1999 interim statement entitled"The Impact of Genetic Modification on Agriculture, Food and Health", the British Medical Association, Board of Science and Education, raised food allergenicity as a key area of concern about genetically modified foods, leading that organization to call for further research on the possible health risks of genetically modified foods consumption. Among other issues, was cited a March 1996 report by US researchers that a major allergen of Brazil nuts (Bertholletia excelsior) had been transferred to soybeans via genetic engineering. The allergen at issue was a high- methionine protein. As a result of this assessment, commercial interest in this transgenic soybean was abandoned (Taylor, 1997). Nonetheless, the case demonstrates the principle that allergens can be transferred between plant species.

An important facet of the overall safety assessment of genetically engineered organisms must be a thorough evaluation for possible allergenic potential. The question addressed is whether the presence of a transgene product alters the allergenic potential of a food or other products interacting with the body surface, by any mechanism. Hence, assessments must be made as to whether the presence of the transgene product unintentionally renders the crop or domesticated animal (or foods processed from them) more allergenic than a nonengineered counterpart.

It would therefore be highly desirable to provide an allergen test that (a) is simple and easy to read, (b) can identify allergenicity of a single heterologous protein, which may be present in low amounts, in a mixture of many other proteins, and (c), distinguish between allergenicity of the protein or allergenicity of the plant material in the transgenic environment. The present invention is designed to most these needs.

Summary Of The Invention Accordingly, it is an object of the invention to provide a method for testing the allergenicity of a heterologous protein produced by genetically modified organism, e. g., a plant or animal that has been genetically modified to produce that protein. In practicing the method, the testing includes the steps of: (a) sensitizing a newborn dog from an atopic dog colony with a first extract prepared from tissue of the genetically modified plant or animal and containing a mixture of plant or animal proteins and the heterologous protein, by injecting, feeding or applying to the skin the extract into the newborn dog; (b) after a period sufficient to allow the dog to establish an immune response to the sensitizing extract, challenging the dog with the extract; (c) observing the degree of allergic response provoked; (d) if a detectable skin reaction is observed, comparing the degree of skin reaction observed with that observed by carrying out steps (a)- (c) above, but where the sensitizing step (a) or applying step (b) is carried out with a second plant or animal extract containing substantially the same proteins as the first extract but lacking the heterologous protein; and (e) if the degree of skin reaction at (c) is greater than that observed by carrying out steps (a)- (c) in accordance with step (d), identifying the heterologous protein as a potential allergen in humans.

The challenging and observing steps may include (i) applying the allergen material to a skin region of the dog and observing a local wheal reaction at the application site as the allergic response (skin test); (ii) feeding the allergen material to the dog, and observing gastrointestinal upset as the allergic response (feeding test); (iii) contacting the allergen material directly with the wall of the stomach of the dog and observing local wheal reaction at the application site as the allergic response (gastroendoscopy test); (iv) administering the allergen material by inhalation to the dog, and observing bronchial constriction as the allergic response (inhalation test); and (v) applying the allergen material with a patch immobilized on the skin and observing inflammation at the site of application.

In a preferred embodiment, the extract is obtained from a transgenic plant. In another preferred embodiment the plant is a crop plant. Preferred crop plants include corn, barley, wheat, rice, peanut, sorghum and soy.

In another embodiment, the comparison of skin reactions observed in step (d) is carried out by applying the first extract to a dog sensitized with said second extract.

In yet another embodiment, substantially no skin reaction is observed in carrying out steps (a)- (c) in step (d). Preferably, the extract can be prepared by forming a tissue powder and extracting the powder with a selected extract medium.

In still another embodiment, the method of testing the allergenicity of a heterologous protein further includes when a potential allergen is identified in step (e), repeating step (c) with the heterologous protein in purified form. In a related aspect, an organism other than the transgenic plant or animal produces the heterologous protein. In another aspect, the transgenic plant or animal produces the heterologous protein.

In one aspect, the method for testing the allergenicity of a heterologous protein further includes, when a potential allergen is identified in step (e), separating proteins in the first extract and reacting the separated proteins with an immunoglobulin obtained from the dog sensitized with the same extract, to identify whether the protein that reacts with the immunoglobulin is the heterologous protein.

In another aspect, the degree of skin reaction observed in step (c), compared with that observed in step (d) is indicative of the degree of allergenicity expected in humans.

It is another, broader, object of the invention to provide a method for testing a biological substance for allergenicity in humans, comprising the steps of: (a) sensitizing a newborn dog from an atopic dog colony with (i) at least one known allergen in humans, (ii) a non-allergen control material, and (iii) a sample containing the test substance, by injecting the allergen, control material, and test substance into the dog; (b) after a period sufficient to allow the dog to establish an immune response to the allergen : (b1) confirming that said sensitizing has provoked an appropriate immune response in the dog by challenging the dog with the known allergen and observing an allergic response in the dog, (b2) confirming that said sensitizing has not provoked an inappropriate immune response in the dog by challenging the dog with the control material and observing the absence of an allergic response in the dog, and (b3) challenging the dog with the test substance and observing the degree of allergic response provoked or no response; and (c) if an allergic response is observed in (b1) and (b3), but not (b2), identifying the test substance as a potential allergen in humans.

In a preferred embodiment, the method is used for grading the degree of allergic response produced by the test material, wherein step (a1) includes sensitizing the dog with at least two different allergens known to provoke a different degree of allergic response in humans, step (b1) includes challenging the dog with each of the at least two different known allergens, thus to determine the degree of immune response associated with the different known allergens, and in step (c) if an allergic response Is observed in (b1) and (b3), but not (b2), matching the degree of response to the test allergen with one or more of the responses observed in step (b1).

In a related embodiment, the known allergens include at least three allergens selected from the group consisting of peanut extract, ragweed proteins, milk proteins, wheat proteins, and soy proteins.

For use in testing a biological substance for allergenicity in humans, a dog is provided that is (i) obtained as a newborn from an atopic dog colony and (ii) sensitized as a newborn with (a) at least one known allergen from humans, (b) a non-allergen control material, and (c) a sample containing the substance to be tested, by injecting the allergen, control material, and test substance into the dog.

In one embodiment, the dog is useful for testing allergens related to a known allergen, wherein the known allergen is a cereal, and the testing allergen is a cereal other than the known allergen.

In a related embodiment, the known allergen is a pollen, and the testing allergen is a pollen other than the known allergen. In another related embodiment, the known allergen is a nut, and the testing allergen is a nut other than the known allergen. In yet another embodiment, the dog is sensitized with at least two different allergens known to provoke a different degree of allergic response in humans.

It is still another object of the invention to provide a composition for use in sensitizing the dog, which includes a mixture of peanut proteins, ragweed proteins, milk proteins, wheat proteins, and soy proteins, in a weight/volume ratio of about 1: 1: 1: 1: 1.

These and other objects and features of the invention will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying tables and figures.

Brief Description Of The Figures Figure 1 is a time course showing the development of sensitivity of atopic dogs to preparations of peanut and ragweed.

Figure 2 is a time course showing the development from birth sensitivity of atopic dogs to preparations of ragweed, milk, soybean and transgenic corn.

Figure 3 shows canine IgE immunoblots with 3 nut preparations obtained with dogs at 1 year of age. Figure 3A displays the IgE response to peanut. Figure 3B demonstrates that sera from the walnut sensitized dogs bound to multiple walnut polypeptides, but not to the large (-7 kD on 13% gels) subunit of Jug r 1, a major human allergen. Figure 3C shows that IgE from the Brazil nut-sensitized dogs specifically bound Ber e 1, the 2S albumin protein large subunit at 7 kD (identified with a closed wedge).

Figure 4 shows directly compares canine IgE reactivity towards the peanut and tree nut extracts with human IgE binding.

Detailed Description Of The Invention I. Definitions The terms below, as used herein, have the following meanings, unless indicated otherwise: As used herein, the term"atopic dog colony"refers to an inbred colony of dogs which demonstrate an IgE-mediated response to common allergens, which can be readily assessed by means of titrated tests including, but not limited to: skin tests, feeding tests, gastroendoscopy tests, inhalation tests, and dermal patch tests.

The term"dermatitis"is intended to mean any of a large family of diseases of the skin that are characterized by inflammation of the skin attributable to a variety of etiologies (Dorland's Medical Dictionary). Dermititis may be caused by inflammation to the skin including endogenous and contact dermatitis such as, but not limited to: actinic dermatitis (or photodermatitis), atopic dermatitis, chemical dermatitis, cosmetic dermatitis, dermatitis aestivalis, and seborrheic dermatitis.

As used herein, the term"transgenic plant"is intended to refer to a plant that has incorporated DNA sequences, including but not limited to genes which are perhaps not normally present, DNA sequences not normally transcribed into RNA or translated into a protein ("expressed"), or any other genes or DNA sequences which one desires to introduce into the non-transformed plant, such as genes which may normally be present in the non-transformed plant but which one desires to either genetically engineer or to have shared expression. The term also includes the progeny of said plant or plant material, including seeds and plant cells. Thus, a plant that is grown from a plant cell into which recombinant DNA is introduced by transformation is a transgenic plant, as are all offspring of that plant that contain the introduced transgene, whether produced sexually or sexually.

As used herein, the term"crop plant"means any edible or non-edible plant grown for any commercial purpose, including, but not limited to the following purposes: cosmetics, seed production, hay production, ornamental use, fruit production, berry production, vegetable production, oil production, protein production, forage production, animal grazing, golf courses, lawns, flower production, landscaping, erosion control, green manure, improving soil health, producing pharmaceutical products/drugs, producing food additives, smoking products, pulp production and wood production. Thus, crop plants include floral plants, trees, and vegetable plants.

As used herein, the term"genetic construct"refers to the DNA or RNA molecule that comprises a nucleotide sequence which encodes the desired protein and which includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells into which it is introduced.

The term"sensitization"is intended for the purpose of this invention to include the induction of acquired sensitivity or of allergy. Likewise, the term"sensitize"is intended for the purposes of this invention to render sensitive or to induce acquired sensitivity.

As used herein,"heterologous DNA"or"heterologous nucleic acid"includes DNA that does not occur naturally as part of the genome in which it is present or which is found in a location or locations in the genome that differs from that in which it occurs in nature.

Heterologous DNA is not naturally occurring in that position or is not endogenous to the cell into which it is introduced, but has been obtained from another cell. Generally, although not necessarily, such DNA encodes proteins that are not normally produced by the cell in which it is expressed. Heterologous DNA can be from the same species or from a different species. Heterologous DNA may also be referred to as foreign DNA. Any DNA that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which is expressed is herein encompassed by the term heterologous DNA. Examples of heterologous DNA include, but are not limited to, DNA that encodes test polypeptides, receptors, reporter genes, transcriptional and translational regulator sequences, or selectable or traceable marker proteins, such as a protein that confers drug resistance.

The terms"heterologous protein","recombinant protein","exogenous protein", and "protein of interest"are used interchangeably throughout the specification and refer to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein. That is, the polypeptide is expressed from a heterologous nucleic acid.

The term"extract'as used herein is intended to mean a concentrate of aqueous soluble plant components from the portion of the plant extracted and can be in aqueous or powdered form.

As used herein, the terms"allergic response"and"immune response"are used interchangeably and refer to an altered reactivity in response to an antigen and manifesting as various diseases, including, but not limited to, allergic rhinitis (seasonal or perennial, due to pollen or other'allergens), asthma, polyps of the nasal cavity, unspecified nasal polyps, pharyngitis, nasopharyngitis, sinusitis, upper respiratory tract hypersensitivity reaction, gastrointestinal reactions and other allergies. Examples of allergies include, but are not limited to, anaphylaxis, allergic rhinitis (seasonal or perennial) or other respiratory allergy, food allergies and atopic skin reactions. Such responses can be Type I that are IgE-mediated immunologic reactions, or they can be Type II or type III that are IgA, IgG or IgM mediated reactions, or Type IV, cellular immune reactions.

The term"observe"is typically used to refer to a visual observation leading to a qualitative or quantitative determination or detection of an allergic response.

The term"organism"relates to any living entity comprised of at least one cell. An organism can be as simple as one prokaryotic cell or as complex as a animal.

"Known allergens"include, but are not limited to, milk, ragweed, wheat, barley, corn, rice, pigweed, soy, peanut, Brazil nut, English walnut, pollen extracts, dustmites, grass pollens, tree pollens (including oak and birch), mugwort, fish, shellfish, cat dander, horse dander, bee venom, wasp venom, and eggs.

As used herein, the term"microbial"includes bacteria, viruses, fungi and other microbes.

II. Method of the invention The invention includes, in one aspect, a method of determining the allergenicity of a heterologous protein contained in a mixture of components that express the protein. It has been discovered that even a minor allergenic component in a complex mixture of potential allergens can be detected. In addition, a determination of whether a transformed organism contains allergens resulting from the transformation process can be made.

Considered below are the steps in practicing the invention.

Plants and animals and other organisms used to produce the heterologous protein can be genetically modified according to standard methods. In general, a selected nucleic acid sequence is inserted into an appropriate restriction endonuclease site or sites in a vector, which is then transformed into cells of the plant or animal. Standard methods for cutting, ligating and transforming, known to those of skill in the art, are used in constructing vectors for use in the present invention. Generally, methods for the creation of genetically modified plants, animals and other organisms for use in practicing the present invention are known to those of skill in the art. See generally, Sambrook, et al., 1989; Ausubel, et al., 1993; and Gelvin, S. B., et al., 1990.

The invention contemplates that the transgenic animals of the invention can be constructed by any of the available methods including pronuclear injection and transfection of embryonic stem cells followed by blastocyst fusion to create chimeric animals. The offspring of the chimeric animals are transgenic animals. Any technique known in the art can be used to introduce the transgene which encodes the heterologous protein into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to pronuclear microinjection (Gordon et al., 1980; Gordon and Ruddle, 1981); retrovirus mediated gene transfer into germ lines (Van der Putten et al., 1985); gene targeting in embryonic stem cells (Thompson et a/., 1989; and electroporation of embryos (Lo, 1983); and sperm-mediated gene transfer (Lavitrano et al., 1989).

A. Dog Colony The method employs a newborn dog of an atopic colony having a number of special characteristics. The dogs in the atopic colony are inbred, and are selected for a genetic predisposition to an allergy. The dogs may have a history of sensitivity to pollens or foods, and can be of a variety of breeds. Preferably, the dogs are spaniels or basenji dogs or mixed breed spaniel/Basenji dogs. However, the dogs are not limited to these breeds. Once the dogs are produced, they can be bred, inbred, crossbred or outbred to produce further atopic colonies for use as dog models according to the present invention.

The dogs have a history of sensitivity to pollens or foods. The sensitivity can be detected using standard immunometric methods to detect serum gE levels in the dog.

These methods include, but are not limited to, IgE immunoblot enzyme linked immunosorbent assays (ELISA), radio-immunoassays (RIA),"sandwich" immunoradiometric assays (IRMA), and enzyme-labeled immunodot assays. Kits for these assays are commercially available from vendors including CMGTIA (Fribourg, Switzerland) and Antibodies Inc. TM (Davis, CA).

Methods for performing an immunodot assay for identifying atopic dogs in accordance with the invention can be found in Ermel et al., 1997. Typically, the immunodot assay involves aliquoting food antigen extracts onto nitrocellulose strips that are then blocked with casein or albumin to prevent nonspecific protein adsorption. The strips are then incubated at 4° for 18 hours in serum from the dog which has been diluted, followed by a 1 hour incubation with a primary anti-canine IgE antibody at room temperature. Bound antibodies can then be detected by incubating with anti-primary antibody immunoglobulins that are coupled to a detectable marker. Examples of suitable detectable markers include but are not limited to: enzymes, coenzymes, enzyme inhibitors, chromophores, fluorophores, chemiluminescent materials, paramagnetic metals, spin labels, and radionuclides. The strips can then be developed and quantitated by standard methods.

As seen in the present invention, dogs sensitized to an allergen from a single source (for example, wheat) can be used for testing allergens from a related source (barley or other cereals). This feature greatly broadens the use of the dog colony for testing foods or other allergenic materials.

B. Sensitizing, challenging, and observing stage B1. Sensitizing : The first step of the method involves sensitizing a newborn dog from an atopic colony with an extract by injecting into, feeding, or applying to the skin, the extract to the newborn dog. An exemplary method for sensitizing newborn dogs is given in Example 2. There are three types of extracts which can be used for sensitizing the dog. i. Test substance/extract: The first type of extract is a test extract, which is prepared from tissue of a genetically modified plant or animal and contains a mixture of plant or animal proteins and a heterologous protein. This extract is alternatively referred to as a test substance. An exemplary method for preparing an extract from a transgenic plant is detailed in Example 1. ii. Control substance/extract : The second type of extract is prepared from tissue of a genetically modified plant or animal and contains a mixture of plant or animal proteins, but lacks a heterologous protein. This extract may alternatively be referred to as a control substance. iii. Known allergens : Finally, the third type of extract that can be used for sensitizing the dog is prepared from known allergens. Examples of known allergens are described in the definitions section above. Examples 1 and 2 provide an exemplary method for preparing extracts of known allergens, including cow's milk, soybean, ragweed pollen, brazil nut and peanut, from commercially available sources.

In one embodiment, the test extract described above is used initially to sensitize the dog. An extract is typically prepared by forming a tissue powder and extracting the powder with a selected extract medium. In one embodiment, the extract is obtained from a transgenic crop plant. Preferred crop plants are corn, barley, wheat, rice, peanut, sorghum, millet, spelt and soy.

According to one embodiment, the heterologous protein is produced by a transgenic plant or animal. According to another embodiment, the heterologous protein is produced by an organism other than a transgenic plant or animal. Examples of such organisms include fungi, bacteria, protozoa, viruses, and algae.

B2. Challenging : The second step of the method involves challenging the dog with the extract after a period sufficient to allow the dog to establish an immune response, and observing the degree of allergic response provoked or no response. The first extract used for challenging the dog is the test extract. An exemplary method showing negligible allergenicity from the extract of genetically engineered corn leaves is given in Example 1. The various methods used for challenging and observing allergic responses in the dog include skin tests, feeding tests, gastroendoscopy tests, inhalation tests and transdermal patch tests. i. Skin test The skin test may be used to challenge the dog by applying the allergen material to a skin region of the dog and observing local wheal formation at the application site as the allergic response. Procedures for skin tests to measure the allergic hypersensitivity reaction are described in Ermel et al., 1997, Buchanan et al., 1997, and del Val et al., 1999. An exemplary method for performing skin tests is given in Example 1. ii. Feeding test The feeding test may be used to challenge the dog by feeding the allergen material to the dog, and observing gastrointestinal upset as the allergic response. Sensitized pups challenged orally with food allergens may respond with clinical manifestations of food allergy including loose"mud-pie"diarrhea, occasional nausea and vomiting. Signs of nausea and vomiting may be acute, observed within 12 hours of food antigen exposure and may be resolved in up to about 4 days. iii. Gastroendoscopy test The gastroendoscopy test is used to challenge the dog by contacting the allergen material directly with the wall or injecting into the stomach of the dog and observing as the allergic response a local wheal at 3 minutes after contact and inflammation at 24 hours after contact at the application site. Procedures for gastroendoscopy tests are described in Ermel et al., 1997.

Generally, on the day before endoscopy the dogs are fed a hypoallergenic liquid maintenance elemental diet. The dogs are premedicated with atropine to minimize gastrointestinal tract secretions during the procedure. Anesthesia can be induced with Telazol (Aveco Co., Inc., Fort Dodge, lowa) to allow intubation. Dogs are positioned in sternal recumbency for the endoscopic examinations.

The endoscopy procedure can be performed with a Pentax upper gastrointestinal tract endoscope (Pentax, Orangeburg, N. Y.) which can be fitted with an ultra miniature endoscopic videocamera. Food antigen extracts are injected into the gastric mucosa via needles passed through the biopsy channel of the endoscope.

Food allergen extracts are administered into the gastric mucosa along the ventral- lateral aspect of the greater curvature of the stomach near the confluence with the pyloric antrum. A series of dilutions of known antigens can be injected into the gastric mucosa to determine the optimal concentration for gastroscopic food sensitivity testing. A mixture of physiologic saline and glycerin can be used as a control. Approximately 5 to 10 minutes before the injections filtered 0.5% (w/v) Evans blue dye solution can be given intravenously to enhance visualization of the allergic response (0.2 ml/kg animal weight).

Gastric mucosal tissue specimens are collected before food extract and control injections with radial jaw biopsy forceps. Gastric mucosal responses are graded according to the amount of swelling, erythema, and blue patching that is observed about 3 minutes after the injection of food extract or control. The injection sites are continuously observed and videotaped for 3 minutes after each injection and biopsy specimens can be obtained immediately after the 3 minute observation period. The injection sites can be re-examined and videotaped at 15 to 30 minutes and 24 to 48 hours after the injections. Additional gastric mucosal tissue specimens are collected from the dogs 24 to 48 hours after injection. The biopsy tissue specimens can be fixed in buffered 10% formalin for histologic examination. The videotapes are reviewed and graded by persons unaware of the identity and order of the injected food antigen extracts. iv. Inhalation test and transdermal patch test The inhalation test may be used to challenge the dog by administering the allergen material by inhalation to the dog, and observing bronchial constriction as the allergic response. A transdermal patch may be used by applying the allergen material with a patch immobilized on the skin and observing inflammation after 24 to 72 hr at the site of application. Both of these methods are standard to one skilled in the art.

C. Analysis of reaction data C1. Simple reaction The third step of the method involves determining whether a detectable skin reaction has been observed after following the first and second steps described above.

C2. Qualitative analysis In one embodiment, if a detectable skin reaction is observed, then the sensitizing, challenging and observing steps carried out above are repeated using a second plant or animal extract containing substantially the same proteins as the first extract but lacking the heterologous protein. The degree of the two skin reactions are then compared to one another.

C3. identifying the source of allergenicity Finally, the fourth step of the invention involves determining whether the heterologous protein is a potential allergen in humans. The protein is identified as a potential allergen in humans if the degree of skin reaction observed following sensitizing and challenging with the first extract is greater than that observed following sensitizing and challenging with the second extract which contains substantially the same proteins as the first extract but lacks the heterologous protein.

In one embodiment, when a potential allergen is identified above, the sensitizing, challenging, and observing steps are repeated with the heterologous protein in purified or partially purified form. An exemplary comparison of the skin test response to transgenic corn leaf extract and the purified protein of interest is shown in Table 2 of Example 1.

In a second embodiment, when a potential allergen is identified above, an additional step which includes separating proteins in the first extract and reacting the separated proteins with an immunoglobulin obtained from the dog sensitized with the same extract, to identify whether the protein that reacts with the immunoglobulin is the heterologous protein.

Standard methods for performing this test include, but are not limited to, enzyme linked immunosorbent assays (ELISA), radio-immunoassays (RIA),"sandwich" immunoradiometric assays (IRMA), and enzyme-labeled immunodot assays. Kits for these assays are commercially available from vendors including CMG (Fribourg, Switzerland) and Antibodies Inc. Tll (Davis, CA).

Standard techniques of protein purification may be employed to separate proteins in the first extract, including : precipitation by taking advantage of the solubility of the protein of interest at varying salt concentrations, precipitation with organic solvents, polymers and other materials, affinity precipitation and selective denaturation; column chromatography, including high performance liquid chromatography (HPLC), ion- exchange, affinity, immuno affinity or dye-ligand chromatography; immunoprecipitation and the use of gel filtration, electrophoretic methods, ultrafiltration and isoelectric focusing.

Each of the above-identified methods is well within the knowledge of the skilled artisan, and no undue experimentation is required to purify the proteins or epitopes of interest from any extract, using the standard methodologies outlined hereinabove, and in the literature.

Example 1 is an exemplary method showing that the invention has the ability to determine whether a transgenic protein of interest is a significant allergen. In this example, two litters of pups were sensitized to a leaf extract of genetically modified corn.

At the same time, pups were sensitized to known allergenic foods, ranging from very strong (peanut) to moderately strong (milk, soy). A pollen allergen extract (giant ragweed) was also included in the sensitization regime as a reference. In parallel, one of the litters was sensitized to barley and another litter was sensitized either to wheat or barley. The results in Example 1 show that corn leaves have negligible allergenicity after being genetically engineered to contain a protein of interest, and that the protein of interest did not become allergenic.

The known allergens used to measure the relative allergenic response in the example were milk, soybean, ragweed, and peanut. However, any known allergen may be used, including those listed in the definitions section above. Likewise, any protein of interest or heterologous protein can be used including, but not limited to, enzymes, receptors, hormones, antibodies or fragments thereof, and growth factors.

Figure 2 is a time course showing the development from birth of sensitivity of atopic dogs to preparations of ragweed, milk, soybean and transgenic corn. Significantly, this figure, along with Example 1, demonstrates that the inbred, highly allergic dogs sensitized to a non-allergenic protein do not exhibit an allergic response when challenged by that protein. Thus, by following the methods described, a determination of whether a transformed organism contains allergens resulting from the transformation process can be made.

D. Use of multiple extracts More broadly, the invention includes a method of testing a biological substance for allergenicity in humans, which includes the step of sensitizing the dog with all three extracts described in section B1 above. Thus, the dog is sensitized with the test substance and at least one known allergen and one known nonallergen. After a period sufficient to allow the dog to establish an immune response, the dog is challenged with each of the extracts used for sensitization, and the allergic response is observed and analyzed as in section C above. If an allergic response is observed following a challenge with a known allergen and with the test substance, but not with the known non-allergen, then the test substance is identified as a potential allergen in humans.

In one embodiment of this broader aspect, the degree of allergic response produced by the test material is graded by sensitizing the dog with at least two different allergens known to provoke a different degree of allergic response in humans and one non-allergen, challenging the dog with each of at least two different known allergens, thus to determine the degree of immune response associated with the different known allergens, and if an allergic response is observed following the challenge with the two different allergens and with the test substance, but not with the control material, then matching the degree of response to the test allergen with one or more of the responses observed in the challenging step with the known allergens.

The known allergens for grading the response are preferably peanut extract, ragweed extract, milk proteins, wheat proteins, and soy proteins.

III. Cross reactivity of related allergens As an important addition, a dog colony may be maintained continuously for testing allergens from a related group, or family of organisms. Example 5 provides an exemplary method of determining cross reactivity of related allergens in the atopic dog model. In this example a population of dogs sensitized to allergens was used to test the allergenicity of related plants.

From the foregoing, it can be appreciated how various objects and features of the invention are met. The testing method of the invention is effective to detect even minor allergenic components in a complex mixture of potential allergens. Accordingly, a determination of whether a transformed organism contains allergens resulting from the transformation process can be made. The following examples illustrate methods of measuring the allergenicity of a protein in accordance with the invention. The examples are intended to illustrate, but in no way limit, the scope of the invention.

Examples Unless otherwise indicated, all reagents and biochemicals were obtained from sources previously identified (Kobrehel et al., 1992).

Example 1 1. Test materials.

Powdered lyophilized leaf material from a transgenic corn plant was extracted with 50 mM Tris-HCI, pH 9.5,0.1 M NaCI, 2 mM EDTA, 2 mM dithiothreitol, 1 mM 4- (2- aminoethl)-benzenesulfonyl fluoride, 1 uM leupeptin. Protein was determined using the Bradford (Bio-Rad) Coomassie blue procedure with albumin as the protein standard.

Concentration of the transgene product was determined by ELISA. The corn plant expresses a transgene protein of interest (POI) : VIP3A. It is an insecticide produced by vegetatively growing Bacillus species. The full name is"vegetative insecticidal protein"as described in Estruch, et al.

This material was found to contain on a percentage basis 0.02% transgenic protein. This extract, designated"transgenic corn leaf extract"or"transgenic preparation" was prepared at a laboratory on the East coast and shipped overnight on wet ice to the University of California, Berkeley.

2. Animals : immunization schedule, transgenic leaf preparation litters.

From the original inbred colony (6 generations) of highly allergic dogs, and breeding resulted in 2 litters (7FB and 7FC, 18 pups), which were immunized with food extracts-commercial preparations of cow's milk (1: 20 w/v), soybean (1: 10 w/v), both from Bayer, a transgenic corn leaf extract (see above) and a commercial giant ragweed pollen extract (1: 20 w/v), also from Bayer. One litter (7FB, 9 pups) was sensitized to a commercial preparation of peanut (1: 10 w/v) from Meridian Biomedical. The allergenic response to the preparations was followed systematically over a two-year period.

At birth, pups were injected subcutaneously with 1 pg of each of these preparations in 0.2 ml alum. A concentration of 1 mg was based on dilution of 1 to 10 or 1 to 20 w/v of the commercial allergen extract. The real protein quantity determined by the Bradford procedure corresponded to 43 to 665 ng protein determined by the Bradford method (Bradford, 1976). At 3,7, and 11 weeks, the animals received subcutaneously 0.5 ml of distemper/hepatitis/parvovirus live vaccine followed in 1 and 7 days after each injection with 1 ug of each allergen in alum, also injected subcutaneously. Subsequently, they were boosted every 2 months with 1 ug each of allergen in alum, then, after one year with 10 ug each of allergen in alum. At 18 months, the animals were given a distemper-hepatitis booster. Pups were injected in their axillae with a mixture containing 0.1 ml (50 to 200 ng based on Bradford) for each commercial allergen preparation or with the maximum allowable amount of the transformed leaf extract (190 ng), which contained 0.036 ng of the transgenic protein. The amount of the test protein is comparable to the level of the allergen bovine serum albumin present in milk used to sensitize the dogs. Bovine serum albumin as an allergen was consistently detected with these animals (del Val et al., 1999).

As shown below, the level of leaf extract elicited an IgE response that was independent of the transgenic protein. Allergens were combined in two groups of three allergens ; one group was injected in each axilla. Commercial alum adjuvant (Mylanta, Johnson & Johnson) (0.1 ml) was added to each of three allergen mixtures. The colony of high IgE-producing atopic dogs was maintained at the Animal Resources Service, University of California, Davis (Ermel et al., 1997). The animals, representing the 7th generation of the colony, were cared for according to the principles in the NIH Guide for the Care and Use of Laboratory Animals.

3. Immunization schedule, wheat, barley, and tree nut allerqens.

One of the litters, sensitized to a leaf preparation from a transgenic plant, (7FB) was also sensitized to barley as described above. The third (7FC) litter of nine pups was differentially sensitized to allergens. Five were sensitized to barley (1: 10 w/v, Bayer) and 4 to wheat (1: 10 w/v, Bayer). Five were also sensitized to English walnut (1: 20 w/v, Bayer) and 4 to Brazil nut (1: 10 w/v, Bayer). The immunization schdule for this litter was as given above. Litter A was not sensitized to the transgenic leaf preparation (7FA) but was sensitized to milk, ragweed, wheat and soy as described above. As indicated, the response to ragweed pollen was compared to birch, oak, and pigweed pollens in ragweed- sensitive dogs. The birch, oak, and pigweed (1: 20 w/v) were obtained from Bayer.

4. Sensitized groups.

After eighteen months, following the distemper-hepatitis vaccination, the thirteen dogs showing a low response to some or all of the allergens in skin tests were removed from the colony. After this change, the dogs showed the following sensitivities.

7FA litter (3 dogs): milk, ragweed, wheat, soy; this litter was not sensitized to transgenic leaf extract and thus can serve as a control.

7FB litter (4 dogs): milk, ragweed, barley, soy, peanut, transgenic leaf preparation.

7FC litter (7 dogs): milk, ragweed, soy, transgenic leaf preparation; (3 dogs): wheat, Brazil nut; (4 dogs): barley, English walnut.

5. Skin tests.

Procedures for skin tests to measure the allergic hypersensitivity reaction have been described elsewhere (Ermel et al., 1997; Buchanan et al., 1997; del Val et al., 1999).

In brief, 0.5% Evans blue dye was injected intravenously 5 minute prior to skin testing.

Aliquots of 0.1 ml of the individual extracts were injected intradermally on ventral abdominal skin in half-log dilutions. Skin reactions were read blindly by the same experienced observer scoring two perpendicular diameters of each blue spot. Appropriate negative controls [diluted in physiological saline (10 mM Na2HP04,1.8 mM KH2PO4, pH 7.4,2.7 mM KCI, 137 mM NaCI)] were included for each animal tested.

6. Results of transgenic leaf preparation tests with allergens of interest.

The results demonstrate that the extract from genetically engineered corn leaves has negligible allergenicity (Table 1). In Table 1 the minimum ng value represents the median amount of the preparation eliciting a wheal for the animals retaining sensitivity for the 23-month period.

As demonstrated by Part A of Table 1, only those dogs giving a response that fell in the range of 10-X or 1/10that of the median response of all dogs tested were included in the calculations. The numbers in parentheses represent the actual number of dogs used for each calculation.

As shown by Part B of Table 1, the relative skin test response of the transformed leaves extract after 23 months was approximately 1/5, ooth that of peanut, a very strong food allergen, to 1/700t"and 1/50th that of the moderate food allergens milk and soybean, respectively, and 1/900th that of ragweed, a well charactized pollen allergen. These values were calculated by dividing the minimal amount of transgenic corn leaf extract giving a wheal at 23 months (65,000 ng) by the minimal amount of the indicated allergen extracts giving a wheal.

Table 1 Development of Alleraenic Response in Dogs : Transgenic Corn Leaf Extract vs.

Known Allergens A. Minimum ng inducing a skin response (wheal > 3mm) Preparation 9 mo 18 mo 23 mo Peanut Avg 10 (4) 22 (4) 12 (4) Std-Dev 10 0 12 Milk Avg 12,594 (7) 337 (8) 77 (7) Std-Dev 17,423 121 135 Ragweed Avg 636 (9) 237 (11) 64 (11) Std-Dev 716 454 76 Soybean Avg 244 (10) 1,195 (10) 1,135 (10) Std-Dev 287 1,908 1,936 Transgenic Avg 112,000 (8) 68,800 (7) 64,960 (9) corn leaf Std-Dev 0 53, 880 55, 820 The minimum ng value represents the median amount of the preparation eliciting a wheal for the animals retaining sensitivity for the 23-month period. Only those dogs giving a response that fell in the range of 10-X or 1/10that of the median response of all dogs tested were included in the calculations. The numbers in parentheses represent the actual number of dogs used for each calculation.

B. Relative Allergenicity Preparation 9 mo 18 mo 23 mo Peanut 11,200 5,091 9,333 (4666) Milk 9 332 1,455 (727) Ragweed 176 473 1,750 (875) Soybean 459 94 99 (50) Transgenic corn leaf 1 2 2 (1) The values were calculated by dividing the minimal amount of transgenic corn leaf extract giving a wheal at 9 months (112,000 ng) by the minimal amount of the indicated allergen extracts giving a wheal.

The results show that, in the dog model, the transgenic protein in the corn leaf is not a significant allergen. This conclusion is confirmed by results shown in Table 2, Part A, which demonstrates that after 18 months exposure to the protein of interest there was no response elicited in dogs sensitized to the transgenic leaf preparation, when tested at levels ranging from 5-X to 380-X the amount present in the parent leaf extract. The quantity of the transgenic corn leaf extract found to be the lowest amount giving a wheal (29 ng) was tested. As indicated in Table 2, the protein of interest was tested using 29 ng protein (approximately 5-X POI), 2.11 pg protein (approximately 380-X POI), and 14.6 ug protein (approximately 2,500-X POI) and 49.2 pg (approximately 7,000-X POI). The number of dogs tested was 11.

Skin tests should detect the protein of interest in the transgenic leaf extract. In work using milk (del Val et al., 1999) it was revealed that 0.10 ng of the milk allergen, serum albumin, was detectable-i. e., about 1/50th the level of the protein of interest tested in the experiment in Table 2, Part A. Only at a level of 7,000-X that present in the transgenic leaf extract was response observed with the protein of interest (18 month trial).

Even at this level, the response was very weak and corresponded approximately to that observed with control corn leaf extract.

7. Relative allergenicity between transaenic corn and purified transaenic protein.

A comparison of the skin test response observed after 9,18, and 23 months for the control and genetically transformed corn leaves is also shown in Table 2. The values in Part A were calculated by dividing the minimal amount of transgenic corn leaf extract giving a wheal, for example, 112,000 ng at 9 months, by the minimal amount of the indicated allergen extracts giving a wheal. The skin test date indicate that there is no significant difference in the allergenicity of the two preparations. The results provide additional evidence that the protein of interest is not an allergen (see also Table 1, Part B).

The data further indicate that the transgenic leaf does not contain allergens resulting from the transformation process.

Table 2 Skin Test Responses to Transgenic Corn Leaf Extract and Different Amounts of Purified Protein of Interest (POI) A. Minimum ng inducing a skin response (wheal ! 3mm) Extract 9 mo 18 mo 23 mo Control corn leaf Avg 88,107 28,305 4,446 Std-47,412 41,464 4,947 Dev Transgenic corn leaf Avg 112,000 68,800 64,960 Std-0 53,880 55,820 Dev POI equivalent to 5-X Avg nr nr nr transgenic corn leaf (29ng) Std---- Dev POI equivalent to 380-X Avg--nr transgenic corn leaf (2.1 Std---- mg) Dev POI equivalent to 2,500-X Avg nr-- transgenic corn leaf (14.6 Std---- mg) Dev POI equivalent to 7,000-X Avg-40, 380 transgenic corn leaf (49.2 Std--13, 590 mg) Dev nr = no reaction The quantity of the transgenic corn leaf extract found to be the lowest amount giving a wheal (29 ug) was tested. As indicated, the transgenic extract was tested using 29 ng protein (approximately 5-X POI), 2.11 Bug protein (approximately 380-X POI), and 14.6 ug protein (approximately 2,500-X POI) and 49.2 ug (approximately 7,000-X POI).

Number of dogs tested: 11 B. Relative Allergenicity Preparation 9 mo 18 mo 23 mo Control corn leaf 1.3 4.0 25.2 Transgenic corn leaf 1.0 1.6 1.7 POI equivalent to 380-X transgenic corn--nr leaf nr = no reaction The values were calculated by dividing the minimal amount of transgenic corn leaf extract giving a wheal at 9 months (112,000 ng) by the minimal amount of the indicated allergen extracts giving a wheal.

8. Course of aller-gen development to known allergens.

The development of the IgE response to the different allergens tested is shown in Figures 1 and 2. Response to peanut, by far the strongest allergen, developed rapidly and was essentially unchanged between the 9 and 23-month tests (Figure 1). By contrast, the response to milk progressed from zero at 9 months to a very significant level at 18 and 23 months (Figure 2). Ragweed followed a similar pattern. Soybean was different and showed the strongest response at 9 months and a weaker response at 18 and 23 months.

Consistent with the data presented above, the transgenic corn leaf preparation showed essentially no response throughout the trial period (Figure 2). The values in Figures 1 and 2 are based on the data in Table 1.

The results indicate that an allergen such as peanut manifests a stable IgE response earlier than the weaker allergens used in this study. Significantly, if this were the case with a test transgenic protein preparation, one could ascertain potential allergenicity for humans as well as dogs in less than one year.

Example 2 1. Dogs.

The animals were descended from a colony of inbred, high IgE-producing spaniel/retriever/Basenji dogs maintained at the Animal Resources Service, School of Veterinary Medicine at the University of California, Davis. The animals, representing the seventh generation of the colony, were cared for according to the principles outlined in NIH publication 85-23, the Guide for the Care and Use of Laboratory Animals. Puppies were nursed for 6 weeks and then weaned to Eukanuba Puppy Small Bites (lams Company, Dayton, OH) as previously described. Ermel, et al. (1997). After 6 months they were fed Eukanuba Puppy Large Bites and at one year of age, Eukanuba Original. An animal health technician and student volunteers trained and socialized the animals.

2. Immunizations.

The puppies were inoculated on day 1 subcutaneously in the axilla with 1 ug of protein diluted as needed in up to 200 jn. L of saline plus 200 ut of alum. The allergen extracts were commercially obtained from Hollister-Stier (Spokane, WA). Protein content was measured by the BioRad protein assay kit (BioRad Inc., Hercules, CA) and then diluted to 1 ug/mL. Dogs were immunized with commercial extracts of peanut (Arachi hypogaea), walnut (Juglans regia) or Brazil nut (Bertholia excels) and either wheat (Triticum aestivum) or barley (Hordeum vulgaris). All dogs were also immunized with cow's milk, giant ragweed (Ambrosia trifida) and soy (Glycine max). At ages 3,7 and 11 weeks, the puppies were vaccinated subcutaneously in the shoulder area with 0.5 mL of live-attenuated distemper-hepatitis vaccine (Pitman-Moor, Washington's Crossing, PA).

Two and nine days after the viral inoculations, the dogs were given the same allergen extracts they had received on the day after whelping. Booster immunizations of the allergens in alum as above were administered bi-monthly in the axilla using 10 ; ng attergen extract.

3. Skin testing As previously described, the ventral aspect of the abdomen was used for intradermal skin testing. The same commercial extracts were used for immunization and skin testing. Approximately 5 minutes before the skin tests, 0.2 mL/kg of filtered 0.5% Evans blue dye (Sigma Chemical Co., St. Louis, MO) was slowly injected intravenously to help visualize the wheal response. Serial dilutions of allergens were prepared fresh daily to determine the minimum concentration of protein needed to induce a wheal response.

The reactions were read at 20 minutes and measured in mm. Commercial negative (saline, Hollister-Stier) controls were placed as well. A positive test was defined as a wheal/flare reaction showing up as a blue area measuring greater than 5 x 5 mm. Animals were tested at 2.5 years of age.

4. IgE Immunoblotting Sera were obtained and used for immunoblotting when animals were 1,2, and, from some of the dogs at 3 years of age. Peanut, walnut and Brazil nut extracts were made in our laboratory by previously published methods. Teuber, et al. (1999) Samples were boiled for five minutes in buffer [60 mM Tris-HCI, pH 6.8,2% SDS, 10% glycerol, 100 mM dithiothreitol (DTT), 0.01% bromophenol blue] and electrophoresis was carried out overnight at 8 mA constant current using a SE600 Vertical Slab Gel Unit (Pharmacia Biotech, Piscataway, NJ). SDS-polyacrylamide gels, 13%, were used for immunoblotting as previously described. Proteins were transferred to 0.22, um nitrocellulose membranes (MSI, Westborough, MA) overnight at 30V using a TE 42 Transphor Electro-Transfer Unit (Pharmacia Biotech, Piscataway, NJ). Nitrocellulose containing the blotted proteins was cut into 3-4 mm wide strips containing approximately 25 p. g of protein per 4 mm strip.

Strips were blocked for 1 hour at room temperature in phosphate buffered saline (PBS)/3% nonfat dry milk/0. 2% Triton X-100 (TX-100). Diluted sera from the immunized dogs or pooled sera from control, non-atopic dogs (1: 5 in PBS/3% nonfat dry milk/0. 2% TX-100) were added to the strips and incubated overnight at 4°C. Additional strips were incubated with PBS/3% nonfat milk/0. 2% TX-100 (buffer control to monitor non-specific binding of the anti-canine IgE polyclonal antibodies). The strips were then washed three times for 20 minutes in PBS/0.01 % TX-100 and incubated overnight with anti-canine IgE- HRP (CMG, Fribourg, Switzerland). Strips were then washed three times in PBS/0.01 % TX-100, and developed with TMB Peroxidase Substrate Kit (Vector Laboratories, Inc., Burlingame, CA). For comparison, strips from the same blots were used for IgE immunoblotting using pooled sera from human patients with a history of anaphylaxis upon ingestion of either peanut, walnut or brazil nut according to previously described methods.

Teuber, et al. (1999). Sera were obtained after informed consent and approval by the institutional review board.

4. Oral challenges.

Dogs were monitored individually in their kennels for 3 days prior to food challenges to ensure normal appetite and the absence of diarrhea or vomiting. Stools were described as firm, semi-soft or runny/loose consistency. Ermel, et al. (1997). On the day of challenge, food was withheld to decrease the chance of gastric torsion. Challenges with the freshly ground nut to which the animal had been sensitized were initiated at 1 gm, followed by 4 gm, 5 gm, and finally 10 gm at 20-30 minute intervals. The total graded challenge dose was thus up to 20 gm. Ground nuts were moistened with water just before challenge to form a slurry, which was placed on the tongue to enable swallowing. Dogs were monitored for vomiting, swelling, naso-ocular signs, pallor of the oral mucosa, lethargy and diarrhea. Direct observation continued for 3 hours after challenges. The dogs were monitored at 5 hours and 7-8 hours, then left over-night with stool/kennel checks three times per day for the next 3 days. If a dog had a significant anaphylactic reaction requiring epinephrine and fluid resuscitation, it was taken to the on-site clinic for treatment and observation. Blood pressure monitoring equipment was not available for these experiments. Challenges were performed in all dogs at 2.5 years of age and again at 3.5 years in selected animals.

6. Immunization Results.

Two litters (B and C) representing the seventh generation of the colony were immunized in the present nut experiments. Four dogs from the 7FB litter were sensitized to peanut: 7FB4,7FB5,7FB6, and 7FB9. Four dogs from a second (7FC) litter were sensitized to walnut : 7FC1,7FC3,7FC4,7FC5 and 3 to Brazil nut: 7FC6,7FC8, and 7FC9. The peanut-and walnut-sensitized dogs were also immunized to barley, and the Brazil nut-sensitized dogs were immunized to wheat. All dogs were immunized to soy.

7. Skin testing.

Dogs were skin tested to nuts at 6 months of age. All of the animals were positive to the commercial extracts used for sensitization. The saline negative controls consistently measured 0 x 0 mm in these animals. Skin tests were repeated at 14 and 26 months of age and the latter results are summarized in Table 3 which shows the minimum ng of protein eliciting a positive skin (wheal). Among the nuts, peanut elicited the strongest response by a wide margin, followed by Brazil nut and then walnut. Peanut and the tree nuts were significantly more potent allergens than soy or the cereals. Among the grains, wheat was the strongest allergen followed sequentially by soy and barley.

Little to no cross-reactivity was seen among the nut preparations. In addition, there was no cross-reactivity between any of the nuts and soy-i. e., although all animals were soy-sensitive, they did not respond to nuts unless sensitized to a particular nut preparation. Additional evidence of the lack of an effect of soy sensitization on sensitization to another nut is seen by the similar minimum doses of soy required to elicit a positive skin test response in the peanut-and tree nut-sensitized dogs (Table 3). The influence of soy immunization on peanut titers is judged to be non-contributory since the walnut and Brazil nut dogs were similarly immunized and showed little to no IgE against peanut. Owing to a closer phylogenetic relationship, there was strong cross-reactivity between the cereals, wheat and barley (Table 4). This finding prompts the question of whether responses of this type occur in humans-a factor of potential significance owing to the common use of barley in infant foods.

8. IaE Immunoblottina Figure 3 shows the canine IgE immunoblots with the 3 nut preparations obtained with dogs at 1 year of age. The blots with sera from the 2-year old dogs showed no significant qualitative differences. Figure 3A displays the tgE response to peanut. The peanut sensitized dogs all showed IgE binding to Ara h 1 at approximately 60 kD (identified with an asterisk) and less extensively to multiple other polypeptides/proteins.

Faint IgE binding is seen to presumed Ara h 2 at 17 and 18 kD (identified with a closed triangle) in 7FB4 and 7FB5 and very faintly in 7FB6 and 7FB9. Based on these immunoblots, Ara h 1 appears to be the dominant protein eliciting an IgE response in the dog. Walnut sensitized dogs showed limited IgE binding to scattered peanut polypeptides, but no response specifically to Ara h 1 or consistently to other peptides. Two of 3 Brazil nut-sensitized dogs (7FC8,7FC9) showed faint IgE binding to polypeptides around 40 kD.

Figure 3B demonstrates that sera from the walnut sensitized dogs bound to multiple walnut polypeptides, but interestingly, not to the large (-7 kD on 13% gels) subunit of Jug r 1, a major human allergen. Teuber, et al. (1998) All dogs showed binding to Jug r 2, the vicilin-like protein at approximately 45 kD (identified with a closed circle).

The peanut sensitized dogs showed some cross-reactivity to multiple proteins and polypeptides, particularly 7FB5, with IgE binding to the presumed 45 kD vicilin-like protein, but this was not accompanied by clinical reactivity (see below).

Figure 3C shows that IgE from the Brazil nut-sensitized dogs specifically bound Ber e 1, the 2S albumin protein large subunit at 7 kD (identified with a closed wedge). All dogs also showed a strong response to an unknown protein of approximately 40 kD (identified with an open triangle). None of the peanut or walnut sensitized dogs showed binding to the low molecular weight Brazil nut proteins, but two peanut dog sera (7FB4,7FB5) and three walnut dog sera (7FC1,7FC3,7FC5) showed reactivity to proteins at approximately 36-37 kD and 47 kD (identified with an open circle). These same dogs resembled two of the peanut-sensitized counterparts (7FB4,7FB5) in showing a response to the unknown 40 kD protein.

Figure 4 directly compares canine IgE reactivity towards the peanut and tree nut extracts with human IgE binding. For peanut, there is general concordance of antigen recognition by IgE except to the lower molecular weight proteins. This is also the case for walnut, where the lack of IgE to Jug r 1 is noted. For Brazil nut, there is complete concordance.

9. Oral challenges.

The question arises as to whether dogs show gastrointestinal symptoms to peanut and tree nuts that accompany the skin test responses. As seen in Table 5, this was the case. Each of the 4 peanut-sensitized dogs reacted upon peanut challenge with vomiting and lethargy within 10 minutes of administering the provocative dose of the allergen preparation. No peanut-sensitized animal exhibited frank cyanosis or collapse.

Angioedema of the lips was not apparent. The provocative dose was 5 gm in one animal, 10 gm in two, and 20 gm in one (respectively, 7FB9,7FB4,7FB5, and 7FB6). Since proteins account for approximately 23% of the weight of peanut, this represents protein doses of approximately 1.2,2.5, and 5 g, respectively. No peanut-sensitized animal reacted clinically to walnut or Brazil nut. All of the peanut-sensitized dogs recovered spontaneously without pharmacologic intervention.

Three of the 4 walnut-sensitized dogs reacted upon walnut challenge at 2.5 years, while the fourth reacted one year later. One dog (7FC3) fed 5 gm total had frank anaphylaxis with cyanosis and vomiting with signs of hypovolemia ; there was no obvious angioedema or wheezing. The dog responded well to epinephrine and intravenous fluids with adjunctive diphenhydramine. Another animal (7FC1) had voluminous diarrhea and lethargy 1.5 hours into the challenge after a total dose of 10 g walnut. The third animal (7FC4) had copious vomiting after a total dose of 20 gm. The fourth dog (7FC5) failed to react to 20 g of walnut at 2.5 years, but when rechecked a year later, had severe a severe vomit response with 1.7 g ground walnut and was given epinephrine and diphenhydramine. None of the walnut-sensitized dogs reacted to challenge with peanut on cross-challenge, but one, 7FC4, was found to have a small vomit in the kennel the morning after challenge with 20 g Brazil nut. Sera from 7FC4 had no visible IgE binding to Brazil nut proteins at either 1 year (Figure 1 C) or 2 years of age (data not shown) and the dog was non-reactive to Brazil nut by intradermal skin test at 26 months of age (Table 3).

Each of the 3 Brazil nut-sensitized dogs reacted to the preparation used for immunization. One (7FC9) had vomiting and lethargy after oral challenge with 1 gm of Brazil nut (approximately 250 mg protein). A second (7FC6) had vomiting after 10 gm, while the third (7FC8) was given an incorrect dose of 20 gm for its initial dose and nearly died with vomiting and hematemesis beginning within 2 minutes of challenge followed by cyanosis and collapse. The dog responded well to epinephrine and intravenous fluids with diphenhydramine and hydrocortisone, but was thereafter extremely anxious when the investigators were doing food challenges. The animal had tolerated 20 gm of walnut prior to the erroneous dose of Brazil nut. Several weeks later, the same dog was given 1 g of peanut, and it promptly vomited. After another break, it was given 1 g peanut, followed by 4 g peanut, which were tolerated. Doses above 5 g were not tried in this dog, owing to its continuing agitation at challenges. The other two Brazil nut sensitized dogs did not react to either walnut or peanut.

Table 3. Sensitivity of allergenic response to peanut and tree nut vs. cereals and soy determined by skin tests. Dogs were sensitized to peanut, tree nuts, barley and wheat as indicated. All animals were sensitized to soy. Sensitization Dogs Peanut Walnut Brazil nut Barley or Soy Wheat (ng inducing a wheal) PeanuVBarley 7FB4 0.02 2,793 74 1,425 6,600 7FB5 0.002 2, 793 7,400 143 660 7FB6 0.2 NR NR 143 66 7FB9 0.002 NR 7,400 143 66 Walnut/Barley 7FC1 222 28 740 1,425 0.66 7FC3 NR 279 740 1,425 66 7FC4 NR 28 NR 14,250 660 7FC5 2,217 279 7,400 NRa 660 Brazil nut/Wheat 7FC6 NR NR 74 177 660 7FC8 22 NR 0. 74 177 660 7FC9 NR NR 7. 4 1, 767 6,600 Mean ng inducing a 0.06 154 27 2,708 (B) 1,518 wheal in sensitized dogs (p<0. 01) b (p<0.01) ° (p<0. 01) d 825 (W) aNon-reactive to a maximum of 142,500 ng injected intradermally bPeanut compared to all others walnut compared to Barley or Soy dBrazil nut compared to Barley or Soy Table 4. Cross-reactivity of cereal allergens in wheat-and barley-sensitized dogs determined by skin test Sensitization Dogs Wheat Barley (ng inducing a wheal) Wheat 7FC6 177 143 7FC8 177 143 7FC9 1,767 1,425 Barley 7FB4 1,767 1,425 7FB5 NA 143 7FB6 17,667 143 7FB9 177 143 7FC1 177 1,425 7FC3 176,667 1,425 7FC4 17,667 14,250 Table 5. Response of peanut-and tree nut-sensitized dogs to oral challenge Sensitization Dogs Walnut Brazil nut Peanut Peanut 7FB4 No reaction No reaction Vomit 20g 20g 10g 7FB5 No reaction No reaction Vomit 20g 20g 10g 7FB6 No reaction No reaction Vomit 20g 20g 20g 7FB9 No reaction No reaction Vomit 20g 20g 5g Walnut 7FC1 Diarrhea No reaction No reaction 10g 20g 20g 7FC3 Cyanosis/collap No reaction No reaction se 5g 20g 20g 7FC4 Vomit Delayed vomit No reaction 20g (>8 hours later) 20g 20g 7FC5 No reaction No reaction No reaction* 20g 20g 20g Brazil nut 7FC6 No reaction Vomit No reaction 20g 10g 20g 7FC8 No reaction Cyanosis/No reaction hematemesis 20g 20g 5g 7FC9 No reaction Vomit No reaction 20b 1g 20g *This animal vomited with 1.7g when tested at 3.5 y 10. Cereal and pollen cross reactivities.

Dogs sensitized to either wheat or barley were tested for cross reactivity to the opposing preparation as well as to corn and rice. The results (Table 6) demonstrate that the dogs sensitized to wheat showed the best cross reactivity. Of 4 wheat sensitized dogs, all were also sensitive to wheat and barley, 2 were sensitive to rice, and one to corn grain. Of the 7 barley-sensitized dogs, 5 showed a significant response to barley, 4 to wheat, 3 to rice, and one to corn. In Part A the minimum ng value represents the minimal amount of the preparation inducing a wheal. In Part B, results were calculated by assuming the response to wheat in each wheat-sensitized dog and to barley in each barley-sensitized dog to be 100%. The data indicate that the wheat dogs are the strongest cross reactors and display very good cross reactivity with barley, some cross reactivity with rice but limited cross reactivity with corn, a grain not considered a major allergen for humans.

Table 6 Cross Reactivity of Cereal Allergens in Wheat and Barley-Sensitized Dogs A. Minimum ng inducing a skin response (wheal > 3mm) Dogs Wheat Barley Corn Rice Wheat 7FA9 177 143 155 680 7FC6-. ;, . 7. :. w 7FC8 177 1, 425 15500* 6800* ? EC9' (, 767 1, 425 Barley 7FB4 1, 767 1, 425 155 680 1FB5..,... 7FB6 I,, 17667* 143 15500* 68000* : TFE £. : !,. t i A 0 nr 0 0 68 7FB6 | 17667* 143 15500* 68000* 07FB9-- 177 ; < 155i Q* 680Z0^*t 7FC1 177 1, 425 nr 680 I 7FC3'17666. 7 l'i425 :, §, 500* 7FC4 17667* 14250* 15500* 68000* B. Relative allergenicity, % Dogs Wheat Barley Corn Rice Wheat 7FA9 | 100 124 114 26 . 7. FCt 10¢ . 0 12j4 0 0.. |. 0 7FC8 100 12 0 0 : 7FC6' 100, 1' Barley 7FB4 81 100 919 209 7FB5 78-1 ; 60 0 2z." : 7FB6 0 100 0 0 81 °. 0 a.,. ........ oo,, 7FC1..... I 8°5,....,. 100.,... 7FC4 0 0 0 0 7FC4 0 0 0 0 7FC4 0 0 0 0 nr = no reaction Numbers with an asterisk (*) indicating a protein concentration > 5,000 ng are insignificant.

A. The minimum ng value represents the minimal amount of the preparation inducing a wheal.

B. For this table, results were calculated by assuming the response to wheat in each wheat sensitized dog and to barley in each barley-sensitized dog to be 100%. The results used were taken from A above.

11. Pollen alleraen cross reactivities.

The animals sensitized to ragweed were tested for a response to other allergenic pollens. Of the 14 dogs tested, 4 showed a significant response (relative allergenicity of at least 0.8%) to pigweed (the closest relative tested) 4 and 3 each to birch and oak tree pollens (Table 7). In Table A of Table 7 the minimum ng value represents the minimal amount of the preparation inducing a wheal, while in Part B, results are calculated by assuming the response to ragweed to be 100%. The results used were taken from Table 7, Part A.

The cross reactivity to ragweed pollen is consistent with the taxonomic relationship among these plants. It is possible that the cross-reactivity among these diverse pollens is at least in part due to profilins, ubiquitous proteins that promote acting polymerization (Valenta et al., 1992). It is known, for example, that the profilin of ragweed pollen is active with IgE elicited by another pollen, viz., mugwort (Hirschwehr et al., 1998) and that allergens in oak and birch pollens cross react with ragweed (Niederberger et al., 1998).

Table 7 Cross Reactivitv of Pollen Allergens in Dogs Sensitized to Ragweed Pollen A. Minimum ng inducing a skin response (wheal 2 3mm) Dogs Ragweed Birch Oak Pigweed Ragweed 7FA2 1590 nr nr nr ZFA8. . 9-nr nr, nr ; 7FA9 1.59 9.1 nr 2.0 7FA9 1. 59 9. 1 nr 2. 0 7FB4 15. 9 9 : 1 9. 8 20. 4- 7FB5 15. 9 910 980 2035 ,.,,., _.. _ _, 7FB60 15 94 nfr, ; nr ; nr 0.;. 9 nr 7FB9 | 0. 159 nr nr 20350* 7FC1 0-'~ Q1 : 59 i 9-10 X 8"., i20 :. 4 7FC3 15. 9 nr nr 20350* 7FC4 ,.'1g,.. nr A nr _ nr ; 9 nr 7FC6 M5. n. ... nr..,... _.., n.} i 7p8 0-C59 nr nr nr 7FO9", 0. x'1, 5, ~ i nr ; nr 0 ; r B. Relative allergenicity, % Dogs Ragweed Birch Oak Pigweed Ragweed 7FA2 : 1 100 0 0 0 loo., out 7FA9 100 17 0 78 0', I7. 5 162 7FB5 100 1. 7 1.6 0. 8 7FBE'100.,'i"° L. ; ° s °,, 7FB9 100 0 0 0 7FC1'l. 00 1. 7 1. 6 0 : 8 0 0 7FC3 | 100 0 0 0 7FC. 00-| °,. °-. >.--° 7FC5 100 0 0 0. 1 7FC6 100 0 0 0., 7FC8w 4.. _-00.. O : O.. O 7FC9 0 1Q0"f 00, 0-; 0 nr = no reaction Numbers with an asterisk (*) indicating a protein concentration > 5,000 ng are insignificant.

A. The minimum ng value represents the minimal amount of the preparation inducing a wheal.

B. For this table, results were calculated by assuming the response to ragweed to be 100%. The results used were taken from A above.

12. Discussion Canine IgE-mediated food hypersensitivity is a relatively common presenting chief complaint in veterinary practice, ranging from atopic dermatitis to nausea/vomiting, diarrhea and anaphylaxis. The information from a dog model of food allergy may, therefore, more closely mimic the situation in humans than rodent counterparts in which allergies do not occur naturally. Evidence for this conclusion is provided by recent studies with wheat Buchanan, et al. (1997) and milk. del val et al. (1999). On the other hand, work by Knippels et al demonstrated that the Brown Norway rat produced IgG and IgE of similar specificity to the human upon oral antigen sensitization without adjuvants, suggesting that more species may evidence spontaneous atopy under the right conditions than previously recognized. Knippels, et al (2000). Miller et al also showed that the Brown Norway rat produced IgE that recognized epitopes similar to human sera with cow's milk antigens. Miller, et al. (1999).

There has been concern that a mouse model of food allergy may have problems due to the ease of induction of tolerance by oral antigen feeding, Houben, et al. (1997), but several investigators have developed promising murine models, including one in DBA/2 mice in which the mice were sensitized orally without adjuvant, [Ito, et al. (1997)]. The use of cholera toxin co-administered orally with antigen has also been effectively used to induce IgE responses. Hanson, et al. (1998); Li, et al. (1999). Li et al also showed that mice sensitized orally with freshly ground peanut developed IgE responses to Ara h 1 and Ara h 2 similar to human, including reactivity with the major human IgE epitopes on Ara h 2. Li, et al. (2000). The dogs described herein were immunized and boosted with commercial preparations rather than freshly prepared extracts-a factor that may explain the lack of a strong response to Ara h 2 and Jug r 1.

Intraperitoneal administration of food antigens in mice, with or without adjuvant, has shown some evidence of a hierarchy response-a feature desirable in an animal model-suggesting a potential application in the evaluation of genetically modified foods.

Dearman, et al. (2000), Evaluation of Allergengenicity of genetically modified foods (2001).

Based on the present and previous [Buchanan, et al. (1997), delVal, et al. (1999)] findings suggestive of a hierarchy response closely resembling the human clinical experience, dogs may also be a potential model to test the allergenicity of genetically modified products. With a small number of dogs, peanut proteins elicited an unprecedented IgE response, followed sequentially by Brazil nut, walnut, soy and then wheat and barley.

Moreover, as in humans, there was no significant cross-reactivity among the nuts.

Relative to peanut, the allergenicity values range from 1/500th with Brazil nut to 1/50, 000th with barley. This hierarchy makes the dog uniquely situated as a model to test the hypothesis that proteins not considered allergenic in humans will only weakly stimulate IgE production, while those known to be potent allergens will be expected to elicit high titers of IgE. This possibility is currently under investigation. It will be of interest to compare results obtained with the present colony to the soft coated wheaten terrier model being developed to test food allergen. (Vaden, et al., 2000).

The dog has additional advantages as a potential model. Its large size permits the performance of gastrointestinal studies, such as sampling of mucosa under endoscopy without sacrificing the animal. Resuscitation of an animal with profound anaphylaxis is also possible (as demonstrated on two occasions during this study) allowing for the possibility of pre-and post immunomodulatory therapy oral challenges without loss of animals used in the initial challenges. Finally, in contrast to other models, which have been successfully sensitized only with single allergens, it is possible to immunize one dog simultaneously with a number of crude food extracts.

The dog is, however, not without a downside. Dogs are expensive to maintain and their immune response is not as well characterized as that of the murine or rat models.

Further, sensitization is a lengthy process, requiring up to 18 months to achieve stable responses.

In summary, a small group of dogs was successfully sensitized to peanut, walnut, Brazil nut, soy, wheat and barley proteins in a manner reflective of the human response, thus strengthening its use as an animal model for the study of food allergy. The observed hierarchy in response to these preparations justifies further testing to determine whether a similar situation applies to other allergens.

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All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains.