Title:  Saliva test for detection of food allergy and intolerance

United States Patent:  6,858,398

Issued:  February 22, 2005

Inventors:  Vojdani; Aristo (Los Angeles, CA)

Assignee:  Immunosciences Lab., Inc. (Beverly Hills, CA)

Appl. No.:  156333

Filed:  May 24, 2002

Abstract

A method for determining the presence of food allergy or food intolerance and their cross-reactive tissue antigens is disclosed. The method includes determining a level of antibodies against a dietary antigen in a mucosal sample from the patient and comparing the level with normal levels of the antibodies. Dietary antigens that were tested include milk and milk products; eggs and egg products; meat and meat products; fish, mollusks, and crustaceans and their products; oils, fats, and their products; grains and grain products; pulses, seed, kernels, nuts, and their products; vegetable and vegetable products; fruit and fruit products; sugar, sugar product, chocolate products, and confectionary; and spices.

Description of the Invention

BACKGROUND OF THE INVENTION

Food allergy has become a problem which concerns many clinicians. Adverse reactions to foods in which the pathogenesis involves an immunological response to food components are appropriately called food-hypersensitivity reactions. This term is considered to be synonymous with "food allergy" (1).

There are only limited data on the prevalence of food hypersensitivity in specific populations. The public's perception of the number of individuals affected by food allergies is far in excess of what can be demonstrated under controlled circumstances. One survey among adult atopic patients revealed that 24% claimed "allergic" symptoms on eating or handling various foods (2). Another survey indicated that concern among family members that one or more of them might have a food allergy made this fear the second most frequent cause of altering the family's dietary intake behind salt restriction for hypertension (3).

Immune-mediated adverse reactions to foods can be divided into distinct clinicopathologic entities based on presentation (immediate or delayed), target organ specificity, and pathogenic mechanisms. By far, the most common reactions are IgE mediated and dependent on activation of mast cells in specific tissues. Such reactions are immediate and in severe cases may be life-threatening. Allergic eosinophilic gastroenteritis in some instances also appears to be due to repeated and frequent IgE-mast cell-mediated reactions in the gastrointestinal mucosal. Food-induced colitis/enterocolitis is observed almost exclusively in infants and children and is not strictly IgE dependent. Finally, gluten-sensitive enteropathy (celiac sprue) and dermatitis herpetiformis are due to abnormal immune responses to gluten (gliadin) that are non-IgE related (4, 5).

Immediate reactions to foods can involve one or more target systems, including the skin, respiratory tract, gastrointestinal mucosal, and cardiovascular system. In a double-blind challenge, the first signs of a reaction usually are noted within minutes following ingestion of a food known to provoke such a reaction, and almost always within one hour. Careful clinical observation has made it possible to document that the signs and symptoms initially follow a pattern reflecting the sites of initial exposure to the incriminated food. Thus, oropharyngeal reactions are frequently reported first, followed by gastrointestinal responses, and then involvement of the skin and respiratory tract (6-7).

The constellation of oropharyngeal symptoms developing simultaneously with food ingestion is termed the oral allergy syndrome. It can occur in the absence of systemic symptoms. This pattern of reactivity to food is most commonly due to fruits and vegetables (7). Symptoms include pruritus, tingling, and angioedema of the lips. Throat tightness, facial flushing, oral mucosal blebs, and hoarseness are reported. The clinical diagnosis of an IgE-mediated immediate reaction to a specific food may be supported by skin testing or by measuring allergen-specific IgE by RAST or ELISA tests (8, 9).

Unlike the immediate effects of IgE-mediated allergy, the IgG and IgA-mediated food allergy and intolerance reactions can take several days to appear. Levels of IgG and IgA antibodies in the blood against different food antigens have been used for demonstration of delayed food allergy and intolerance reactions. Therefore, raised serum or plasma IgG and IgA levels of food-specific antibodies are often associated with food allergies. However, measurement of IgG or IgA in the blood may miss abnormal immune reaction to many food antigens (10-13). In one instance, it is known that oral or intragastric administration of dietary soluble proteins such as bovine gammaglobulin (BGG) and ovalbumin or eggalbumin results in salivary IgA production, but not in any antibody production in serum (14-16).

Manifestation of Antibodies

The deposition of antigens in the gut has been shown to lead to the production of IgA antibodies in secretion at sites distant from the gut, such as colostrums, lacrimal and salivary secretions in man and salivary secretions in rhesus monkeys and in rats (17-19).

A general conclusion, therefore, is that the secretory immune system can be stimulated centrally and that precursors of IgA-producing cells migrate from the gut-associated lymphoid tissue to several secretory sites in addition to the lamina propria of the gut itself. Therefore, if antigens are injected into the submucosal tissues, they are likely to induce serum IgG antibodies as well as secretory IgA antibodies in saliva. However, if it is applied topically to the skin or to the intraepiethelial tissue, secretory IgA is the main product, which is detected in saliva. The role of topically applied antigen in the localization and persistence of IgA responses has been demonstrated in several secretory sites, including the respiratory tract, oral cavity, gut and vagina (20-22).

The evidence that cells migrate from the gut to various secretory tissues, and that immunization in the gut leads to antibodies at various secretory sites has led to the concept of a common mucosal system. However, this concept may be an oversimplification, since although immunization in the lung may lead to antibodies in distant secretory sites, such as salivary glands, immunization in the lacrimal glands has also been shown to lead to the production of antibodies in saliva. Thus, with firm evidence that antigen deposition in the gut may lead to antibodies not only in the gut but also in saliva, lungs, lacrimal secretions and genitourinary tract, it is probably more correct to designate the system as an enteromucosal system (2, 3).

Saliva is a source of body fluid for detection of an immune response to bacterial, food, and other antigens present in the oral cavity and gastrointestinal tract. Indeed, salivary antibody induction has been widely used as a model system to study secretory responses to ingested material, primarily because saliva is an easy secretion to collect and analyze.

It seems to be a general feature that salivary IgA antibodies can be induced in a variety of species in the absence of serum antibodies. This has been demonstrated after immunization with particulate bacterial antigens in human could selectively induce an immune response to Streptococcus mutans by oral administration of the antigen. This route of administration resulted only in antibody production in saliva and not in serum. Similar mucosal immune response in the form of saliva IgA did occur in monkeys, rabbits, rats, and mice after oral administration of Streptococcus mutans, Staphylocuccus or different viral antigens and peptides (13, 14-23). The lack of production of IgG, but IgA production in saliva after oral or intragastric administration of bacterial antigens is shown in the following Table 1.

Secretory IgA is capable of functioning as a blocking antibody, which can create a barrier to certain macromolecules, bacteria, and viruses. The interaction with secretory IgA will not permit such antigens to interact with the mucosa and blocks their entrance and exposure to the gut-associated lymphoid tissue. This blockage permits the host to shield efficiently the systemic immune response, local immune response, or both, from being bombarded by many molecules.

The properties of human IgA in serum and saliva are completely different. Serum IgA is monomeric and contains 80-90% IgA₁ and 10-20% IgA₂ while secretory IgA is polymeric and contains 50-75% IgA₁ and 25-50% IgA₂.

Because of these properties, secretory IgA can bind to the invading organisms more effectively. Therefore, secretory IgA have anti-bacterial, anti-fungal, and anti-viral activities, and play an important role in protection of mucosal surfaces from adherence of microorganisms. This prevention of colonization of the mucous membrane by secretory IgA is done by binding and blocking of specific binding sites on the bacterial cell wall. A decrease in adherence results in enhanced clearance of the bacteria by oral secretion and immunological mechanisms. For this reason in patients with secretory IgA deficiency, frequent infections have been observed.

An additional role of secretory IgA is prevention of diffusion of food antigens into mucous membranes. Therefore, a secretory IgA deficient person is more exposed to high levels of antigens or allergens. This phenomenon, along with T-cell regulatory abnormalities which occurs in most patients with IgA deficiency, make them more prone to development of allergies and autoimmune diseases.

Despite the enteric route of exposure to food antigens and peptides, food-specific antibodies are measured only in blood, but not in saliva.

SUMMARY OF THE INVENTION

For the above reasons, saliva has been selected not only because of its relevance in disease, but mainly because it is an accessible fluid, easy to collect, and is thought to show representative responses in secretions after enteric or intragastric immunization. Disclosed herein is a method of measuring salivary IgA and IgM against different food antigens and peptides for use in determining food allergy and food intolerance.

A method for determining the presence of food allergy or food intolerance in a patient includes (a) determining a level of antibodies against a dietary antigen present in the food in a mucosal sample from the patient; and (b) comparing the level determined in step (a) with normal levels of the antibodies in said mucosal sample.

The possible outcomes for the comparison include (i) lower than normal levels or about normal levels of dietary antigen antibodies indicate optimal conditions; and (ii) higher than normal levels of dietary antigen antibodies indicate a food allergy or food intolerance.

In another embodiment, there is a method for determining a type of antibody in a presence of food allergy or food intolerance to a food in a patient, comprising (a) determining a level of antibodies against a dietary antigen present in the food in a first mucosal sample from the patient; (b) determining a level of antibodies against cross-reactive tissue antigen present in a second muscosal sample from the patient, wherein said first and second samples are the same or different; and (c) comparing the level of antibodies determined in steps a) and b) with normal levels of the antibodies.

The possible outcomes for the comparison include (i) about normal levels of antibodies against the dietary antigen and normal levels of antibodies against cross-reactive tissue antigen indicate optimal conditions; (ii) higher than normal levels of antibodies against the dietary antigen and about normal levels of antibodies against cross-reactive tissue antigen indicate food allergy and intolerance without cross-reacting to tissue antigens; (iii) about normal levels of antibodies against the dietary antigen and higher than normal levels of antibodies against cross-reactive tissue antigen indicate an autoimmune reaction not related to the dietary antigens; and (iv) higher than normal levels of antibodies against the dietary antigen and higher than normal levels of antibodies against cross-reactive tissue antigen indicate a presence of food allergy and intolerance resulting in an autoimmune reaction.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A single test that will accurately inform a physician of clinical conditions used to diagnose patients who may suffer from food allergies or food intolerance has been developed. The test utilizes a test method that measures antibody titers to dietary antigens. The test can also utilize a test method that measures the antibodies' ability to bind to a recombinant antigen, synthetic peptide, a peptide prepared by enzymatic digestion corresponding to the dietary antigen, or different cross-reactive tissue antigens.

In order to assist the physician to make a more etiologic based diagnosis, we have developed an immunoassay for detecting food allergies and food intolerance in a patient using mucosal secretions. Mucosal secretions are secretions of a mucosa, such as saliva. Although saliva is mentioned throughout the disclosure, it is not meant to be limiting and can refer to any mucosal secretion.

The test involves using a method for detecting food allergies and food intolerance in a patient. The method includes (a) determining a level of antibodies against a dietary antigen in the food in a mucosal sample from a patient; and (b) comparing the level determined in step (a) with normal levels of the antibodies in said mucosal sample.

The possible outcomes for the comparison include (i) lower than normal or about normal levels of dietary antigen antibodies indicate optimal conditions; and (ii) higher than normal levels of dietary antigen antibodies indicate a food allergy or food intolerance.

As mentioned previously, food allergy has become a problem which concerns many clinicians. Adverse reactions to foods in which the pathogenesis involves an immunological response to food components are appropriately called food-hypersensitivity reactions. This term is considered to be synonymous with "food allergy". Food intolerance is an adverse reaction to food or food components which does not involve the immune system. Food intolerance can be caused by a metabolic reaction to an enzyme deficiency, such as the inability to digest milk properly; by food poisoning, such as ingesting contaminated or spoiled fish; or a food idiosyncrasy, such as sulfite-induced asthma.

Dietary antigens of the preferred embodiments are classified into the general groups as followed: milk and milk products; eggs and egg products; meat and meat products; fish, mollusks, and crustaceans and their products; oils, fats, and their products; grains and grain products; pulses, seeds, kernels, nuts, and their products; vegetables and vegetable products; fruits and fruit products; sugar, sugar products, chocolate products, and confectionary; and spices and herbs.

"Milk and milk products" include, but are not limited to, American cheese, cheddar cheese, cottage cheese, cow's milk, goat's milk, Swiss cheese, and yogurt.

"Eggs and egg products" include, but is not limited to, eggs.

"Meat and meat products" include, but are not limited to, beef, chicken, pork, and turkey.

"Fish, mollusks, and crustaceans and their products" include, but are not limited to, clam, codfish, crab, halibut, lobster, oyster, salmon, sardine, scallop, shrimp, sole, trout, and tuna.

"Oils, fats, and their products" include, but is not limited to, butter.

"Grains and grain products" include, but are not limited to, barley, buckwheat, malt, oat, rice, rye, and wheat.

"Pulses, seeds, kernels, nuts, and their products" include, but are not limited to, almond, cashew, coffee, cola nut, lima bean, millet, peanut, pinto bean, safflower seed, sesame, soybean, sunflower seed, and walnut.

"Vegetables and vegetable products" include, but are not limited to, broccoli, cabbage, carrot, cauliflower, celery, corn, cucumber, eggplant, green pea, green pepper, iceberg lettuce, mushroom, onion, potato, spinach, squash, string bean, sweet potato, and tomato.

"Fruits and fruit products" include, but are not limited to, apple, avocado, banana, blueberry, cantaloupe, grape, grapefruit, lemon, olive, orange, peach, pineapple, and strawberry.

"Sugar, sugar products, chocolate products, and confectionary" include, but are not limited to, chocolate, honey, and cane sugar.

"Spices and herbs" include, but are not limited to, chili powder, cinnamon, garlic, mustard seed, parsley, tea, and yeast.

"Cross-reactive tissue antigens" include, but are not limited to, lectins, lectins receptors, tropomyosin, smooth muscle, epitheleal cell antigens, enzymes, cytochrome P-450 enzymes, transglutaminases, and others.

Food allergy or food intolerance can result in significant level of antibodies against the dietary antigen. The antibodies are present as saliva IgA or IgM. Saliva can be a source of body fluid for the detection of immune response to dietary antigens present in the nasopharyngeal cavity and gastrointestinal tract and can be obtained with relative ease from a patient. The salivary antibody induction has been widely used as a model system to study secretory responses to ingested material in a patient, primarily because saliva is an easy secretion to collect and analyze.

The detection of antibodies can be performed with an immunoassay. Immunoassays include, but are not limited to, ELISA test, RIA test, latex agglutination, beads assay, and proteomic assays. A preferable immunoassay is the ELISA test. Other immunoassays can be used and the choice of immunoassay can be determined by one of ordinary skill in the art.

A normal reading is derived from a baseline measurement taken from antibody measurements for individuals without symptoms relating to food allergies or food intolerance. A baseline measurement for the test is obtained by observing the antibody measurements for individuals without symptoms relating to food allergies or food intolerance. For example, most readings for antibody measurements from an individual without symptoms relating to food allergies or food intolerance are below a certain reading. Preferably, about 50-100% of the readings from an individual without symptoms relating to food allergies or food intolerance is below the certain reading, more preferably about 60-100%, 70-100%, or 80-100% of the readings, even more preferably about 90-100% of the readings. Hence, preferably, if an individual exhibits antibody measurement two standard deviations above the baseline, the above-normal antibody measurement indicates presence of food allergy or food intolerance.

Additionally, antibodies against cross-reactive tissue antigens may be tested. Cross-reactive tissue antigens include, but are not limited to, lectins, lectins receptors, tropomyosin, smooth muscle, epithelial cell antigens, enzymes, cytochrome P-450 enzymes, and transglutaminase. Ingested dietary antigens may induce antibodies that react with the specific dietary antigen and another antigen, such as a cross-reactive tissue antigen. If antibodies against cross-reactive tissue antigens are tested in addition to the dietary antigens, then the antibodies can be determined to be protective or pathogenic.

About normal levels of antibodies against the dietary antigen and normal levels of antibodies against cross-reactive tissue antigen indicate optimal conditions. Higher than normal levels of antibodies against the dietary antigen and about normal levels of antibodies against cross-reactive tissue antigen indicate food allergy and intolerance without cross-reacting to tissue antigens. About normal levels of antibodies against the dietary antigen and higher than normal levels of antibodies against cross-reactive tissue antigen indicate an autoimmune reaction not related to the dietary antigens. Higher than normal levels of antibodies against the dietary antigen and higher than normal levels of antibodies against cross-reactive tissue antigen indicate a presence of food allergy and intolerance resulting in an autoimmune reaction.

Claim 1 of 21 Claims

What is claimed is:

1. A method for determining a presence of food allergy or food intolerance to a food in a patient, comprising:

(a) determining a level of antibodies against a dietary antigen present in the food in a saliva sample from said patient;

(b) comparing the level determined in step (a) with normal levels of said antibodies in said saliva sample, wherein

(i) lower than normal levels or about normal levels of dietary antigen antibodies indicate optimal conditions;

(ii) higher than normal levels of dietary antigen antibodies indicate a food allergy or food intolerance to the food.

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