WO2003046563A1

WO2003046563A1 - Food intolerance immunoassay

Info

Publication number: WO2003046563A1
Application number: PCT/GB2002/005194
Authority: WO
Inventors: Peter Cousins
Original assignee: Yorktest Laboratories Limited
Priority date: 2001-11-27
Filing date: 2002-11-19
Publication date: 2003-06-05
Also published as: AU2002339184A1; GB0128310D0

Abstract

The invention relates to an immunoassay for the detection of antibodies which bind food allergents the presence of which is linked to food intolerance.

Description

FOOD INTOLERANCE IMMUNO ASSAY

The invention relates to an imrnunoassay for the detection of antibodies which bind food allergens the presence of which is linked to food intolerance.

A number of chronic pathological conditions are thought to be caused or provoked by allergic reactions to allergens present in food. For example, conditions such as irritable bowel syndrome, migraine, eczema, arthritis, asthma, autism, Candidiasis, celiac disease, chronic fatigue, diabetes, ear infections, fϊbromyalgia, hyperactivity, hypoglycemia, hypertension, leaky gut, skin rashes and, sinusitis, are each considered in some cases to be provoked or caused by intolerance to allergens present in food.

The following will illustrate conditions caused, at least in part, by food intolerance.

Irritable bowel syndrome (IBS) is a chronic bowel disorder resulting from abnormal contractions of the large intestine. These contractions lead to spasms in the colon causing abdominal pain and irregular bowel movements. The symptoms can last for several days or even months. Research into the causes of IBS have not discovered any anatomical or biochemical abnormalities in the bowel of sufferers of IBS nor are there any specific tests to determine whether a person, or animal, is susceptible to IBS. The condition is not life threatening but can cause debilitation resulting in hospitalisation and absenteeism from work. It is thought that diet has an influence on the severity of IBS and that stress can also contribute.

A further example of a condition thought to be provoked or caused by food allergy is migraine. The exact cause of migraine is uncertain but is likely to be multi-factorial. A theory currently favoured is that those suffering from the condition have inherited a more sensitive nervous system response than those who do not suffer from migraine. During a migraine attack changes in the brains activity produce inflamed blood vessels around the brain. It is known that certain triggers provoke a migrane attack. The diet of a migraine sufferer is thought to be a major trigger of migraine. These foods include alcohol, especially red wine, foods containing monosodium glutamate, and food containing tyramine (eg aged cheeses, preserved meats with nitrates and nitrites). Other factors thought to be involved include: disturbed sleeping patterns; irregular fluctuations in hormone levels (women may have attacks correlated with their menstrural cycle); stress and anxiety; and environmental factors such as weather, fluorescent lights, computer screens, strong odors and high altitude. Clearly migraine is caused by a combination of factors which are not easily controlled.

Food allergy occurs when the immune system (a combination of immune cells, antibodies and chemical mediators) reacts to an allergen present in food to remove it from an animal's system. Currently there are a number of food allergy test kits which screen an individual against a large number of potential food allergens. These test kits are expensive and require the patient to visit a hospital or doctors surgery so that a sample of blood may be taken and tested. It would be desirable if a preliminary screen could be conducted to determine if the patient would benefit from the broader screen. The preliminary screen would ideally be conducted in the home and would comprise testing a blood sample against a narrower combination of allergens which would be indicative of food intolerance. The test would be a simple positive or negative and, if positive, would encourage the patient to pay for a more expensive and extensive test to determine the specific foods which the patient should avoid.

According to a first aspect of the invention there is provided a composition comprising a combination of allergens wherein said combination is a mixture of proteins derived from wheat, milk and egg white.

In a preferred embodiment of the invention said wheat proteins are selected from the group consisting of: gliadins and/or; glutenins and/or; albumins and/or; and/or globulins. hi a further preferred embodiment of the invention said milk protein is casein. Preferably said casein is derived from cows milk.

In a further preferred embodiment of the invention said egg protein is ovalbumin.

h a further preferred embodiment of the invention the combination comprises between 10% and 15% (w/v) wheat protein, preferably about 14%(w/v).

In a further preferred embodiment of the invention said combination comprises between 65% and 75% (w/v) casein, preferably about 69% (w/v).

hi a further preferred embodiment of the invention said combination comprises between 15 and 20% (w/v) ovalbumen, preferably about 17% (w/v).

i a further preferred embodiment of the invention said combination consists of the following combinations of allergens: 14%(w/v) wheat protein: 69%(w/v) casein: 17%(w/v) ovalbumen.

an alternative preferred embodiment of the invention said composition comprises wheat protein at a concentration of between about 0.25- 0.5 mg/ml. Preferably, the wheat proteins and are provided at a concentration of about 0.4mg/ml.

In a further alternative preferred embodiment of the invention said composition comprises milk protein at a concentration of between about 0.5-1.5mg ml. Preferably said milk protein is casein at about lmg/ml.

In a further alternative preferred embodiment of the invention said egg protein is provided at between about 50-150 mg/ml. Preferably said egg protein in ovalbumen provided at about lOOmg/ml. In a further preferred embodiment of the invention said composition comprises the following: wheat protein at about 0.4mg/ml; casein at about lmg/ml; and ovalbumen at about lOOmg/ml.

In a further preferred embodiment of the invention said composition further comprises a buffer or diluent.

In a yet further preferred embodiment of the invention the components of the allergen composition are associated, coupled or crosslinked to a detectable label. Preferably said detectable label is biotin.

The present application has determined the identity of the major food allergens implicated in food intolerance. Those individuals with a positive reaction to this combination of allergens would benefit from a more extensive test screen against a broader range of food allergens to determine the identity of specific food groups which should be avoided. Moreover the present application has determined the optimal ratio of the components used in the initial screen to determine an individuals sensitivity.

According to a further aspect of the invention there is provided a substrate to which is applied, coupled or crosslinked, to at least part of said substrate, an allergen composition comprising wheat extract, cows milk and egg white.

In a preferred embodiment of the invention said composition comprises wheat extract, casein and ovalbumen.

In a further preferred embodiment of the invention the components of said composition are provided at a concentration sufficient to saturate all of the binding sites on said substrate, typically between 5.0mg/ml and 15 mg/ml. According to a further aspect of the invention there is provided a method to test whether an animal is intolerant to at least one food allergen comprising the steps of:

i) contacting a body fluid sample with the composition according to the invention; ii) measuring or detecting the binding of antibodies in said body fluid sample with the allergens comprising the composition.

In a preferred method according to the invention the composition in (ii) is applied to the substrate according to the invention.

In a preferred method of the invention said body fluid sample is selected from the group consisting of: blood/serum; semen; lymph fluid; cerebrospinal fluid; synovial fluid; tears; sweat; urine; saliva; or bone marrow. Preferably, said body fluid sample is blood or serum. Typically the patient directly provides the body fluid sample without the need to visit a medical practioner.

The detection of antibody: antigen complexes is well known in the art. Typical methods involve the detection of an antibody: antigen complex using a labelled secondary antibody directed to the antibody bound to the antigen. The secondary antibody can be labelled with an enzyme (eg horse radish peroxidase; alkaline phosphatase) or a fluorescent label (eg fluoresceine, rhodamine) or with gold particles. Alternatively, the antibodies present in the serum can be directly labelled followed by incubation with the allergen. This type of assay is referred to as an Enzyme Linked ImmunoSorbant Assay (ELISA) or Enzyme Linked hnmunoassy (ELA).

A preferred method according to the invention is the use of the so-called sandwich immunoassay. This involves mixing a body fluid sample with a biotin labelled food allergen and with gold-labelled avidin. The avidin has a high affinity for the biotinylated allergen. Bivalent antibodies which bind the biotin: avidin allergen complex present in the body fluid sample bind to the allergen present on the test substrate. The gold label serves as a visualisation agent. The sandwich method provides for a sensitive assay for the presence of allergen specific antibodies since only when the antibody forms a bridge between the biotin: allergen and the avidin: gold is a positive result obtained.

In a further preferred method of the invention said method detects an immunoglobulin. Preferably said immunoglobulin is selected from the following Ig isotypes: IgA, IgM, IgD, IgE and IgG.

hi a yet further preferred method of the invention said immunoglobulin is IgG. Preferably said IgG is selected from the group consisting of: IgGl, IgG2, IgG3 or IgG4.

Antibodies, also known as immunoglobulins, are protein molecules which have specificity for foreign molecules (antigens). Immunoglobulins (Ig) are a class of structurally related proteins consisting of two pairs of polypeptide chains, one pair of light (L) (low molecular weight) chain (K or λ), and one pair of heavy (H) chains (γ, α, μ, δ and ε), all four linked together by disulphide bonds. Both H and L chains have regions that contribute to the binding of antigen and that are highly variable from one Ig molecule to another, hi addition, H and L chains contain regions that are non-variable or constant.

The L chains consist of two domains. The carboxy-terminal domain is essentially identical among L chains of a given type and is referred to as the "constant" (C) region. The amino terminal domain varies from L chain to L chain and contributes to the binding site of the antibody. Because of its variability, it is referred to as the "variable" (V) region.

The H chains of Ig molecules are of several classes, α, μ, σ, α, and γ (of which there are several sub-classes). An assembled Ig molecule consisting of one or more units of two identical H and L chains, derives its name from the H chain that it possesses. Thus, there are five Ig isotypes: IgA, IgM, IgD, IgE and IgG (with four sub-classes based on the differences in the H chains, i.e., IgGl, IgG2, IgG3 and IgG4). Further detail regarding antibody structure and their various functions can be found in, Using Antibodies: A laboratory manual, Cold Spring Harbour Laboratory Press.

In a further preferred method according to the invention said body fluid sample is combined with a biotinylated food allergen to which is further added avidin which is provided with a detectable label. Preferably said label is gold.

According to a further aspect of the invention there is provided a kit comprising: an allergen composition according to the invention; a substrate according to the invention; detection means for the detection or measurement of allergen: antibody complexes; buffers and cofactors.

According to a further aspect of the invention there is provided an immunoassay as herein described with reference to the description and drawings.

An embodiment of the invention will now be described by example only and with refererence to the following figures:

Figure 1 illustrates the lateral flow device; and

Figure 2 illustrates a summary of the sandwich assay utilising the lateral flow device.

Materials and Methods

Preparation of the test line A mixture of casein (Sigma, UK), ovalbumin (Sigma) and wheat (Antigen Laboratories Inc., USA) is diluted in 50mM carbonate buffer, pH9.6 at lmg/ml, lOOmg/ml and 0.4 mg/ml wheat extract, respectively.

Using an automated striping system (Biodot XYZ Platform Line Sprayer), test lines containing the allergens in carbonate buffer are sprayed onto a nitrocellulose lateral flow membrane card 12mm from the top of the card at lμl/cm .

Preparation of biotinylated proteins

Biotinylated casein is prepared from a starting concentration of 2mg/ml using an EZ- Link™ Silfo-NHS-Biotinylation kit (Pierce, UK). Biotinylated ovalbumin is prepared from a starting concentration of lOmg/ml using an EZ-Link™ Silfo-NHS- Biotinylation kit (Pierce, UK). Biotinylated wheat is prepared from crude wheat flour at a starting concentration of 2mg/ml by Immunoselct GmbH (Germany).

Biotinylated casein is diluted 1/100, biotinylated ovalbumin is diluted 1/400 and biotinylated wheat is diluted 1/1000. All are diluted in Phosphate buffered saline, pH7.2. Biotinylated proteins are mixed 1:1:1 and used to soak a conjugate release pad ^* at the equivalent volume of 15μl per single 2.5mm strip. Conjugate release pads are applied to the nitrocellulose.

a conjugate release pad comprises materials which have a open glass fibre structures. They are designed to allow immunogold and biotinylated proteins to be dried down onto a solid phase substrate. They have an open structure to allow rapid penetration of the pad by the sample and maximum conjugate release.

Preparation of avidin-labelled gold

Avidin-labelled 40nm colloidal gold particles (OD10) prepared by British Bio-cell International (UK) are diluted to OD2 in Tris buffered saline/l%BSA/0.1% Tween- 20 (TBSBT). The OD2 avidin-labelled gold is used to soak a conjugate release pad at the equivalent volume of 30μl per single 2.5mm strip. Conjugate release pads are applied to the nitrocellulose strips along with a serum separating material. The separating material is a membrane prepared from a polymeric blend solution containing mainly two polymers, a hydrophobic polyerthersulphone (PES) and a hydrophilic polyvinylpyrrolidone (PVP) The strips are then cut to 2.5mm width.

Running the assay

A small pinprick of blood (50μl) is applied to a sample application pad along with 30μl of TBSBT. The test is complete within 45 minutes where a positive result shows a pink line at test line height and a negative result does not. False positive results can occur after 2 hours so the test should be read within 90 minutes to be valid.

Claims

1. A composition comprising a combination of allergens wherein said combination is a mixture of proteins derived from wheat extract, milk and egg white.

2. A composition according to Claim 1 wherein said wheat proteins are selected from the group consisting of: gliadins and/or; glutenins and/or; albumins and/or; and/or globulins.

3. A composition according to Claim 1 or 2 wherein said milk protein is casein.

4. A composition according to any of Claims 1-3 wherein said egg protein is ovalbumin.

5. A composition according to any of Claims 1-4 wherein said combination comprises between 10% and 15% (w/v) wheat protein.

6. A composition according to Claim 5 wherein said wheat protein is about 14%(w/v).

7. A composition according to any of Claims 1-6 wherein said combination comprises 65% -75% (w/v) milk protein.

8. A composition according to Claim 7 wherein said milk protein is about 69%

(w/v).

9. A composition according to any of Claims 1-8 wherein said combination comprises 15-20% (w/v) egg protein.

10. A composition according to Claim 9 wherein said egg protein is about 17% (w/v).

11. A composition according to any of Claims 6, 8 or 10 wherein said composition consists of the following combinations of allergens: about 14%(w/v) wheat protein: about 69%(w/v) milk protein: and about 17%(w/v) egg protein.

12. A composition according to any of Claims 1-4 wherein said composition comprises wheat protein at a concentration of between about 0.25-0.5 mg/ml.

13. A composition according to Claim 12 wherein the wheat protein is provided at a concentration of 0.4mg/ml.

14. A composition according to any of Claims 1-4 or 12 or 13 wherein said composition comprises milk protein at a concentration of about 0.5 - 1.5 mg/ml.

15. A composition according to Claim 14 wherein said milk protein is casein at about lmg/ml.

16. A composition according to any of Claims 1-4 or 12-15 wherein said egg protein is provided at between about 50-150 mg/ml.

17. A composition according to Claim 16 wherein said egg protein in ovalbumen provided at about 100 mg/ml.

18. A composition according to any of Claims 12-17 wherein said composition comprises the following: wheat protein at about 0.4mg/ml; casein at about lmg/ml; and ovalbumen at about 100 mg/ml.

19. A composition according to any of Claims 12-18 wherein said composition further comprises a buffer or diluent.

20. A composition according to any of Claims 1-19 wherein the allergens are associated, coupled or crosslinked to a detectable label.

21 A composition according to Claim 20 wherein said label is biotin.

22. A composition according to Claim 20 or 21 wherein said label is coupled or crosslinked to gold.

23. A substrate to which is applied, coupled or crosslinked, to at least part of said substrate a composition comprising wheat extract, cows milk and egg white.

24. A substrate according to Claim 23 wherein said composition comprises wheat extract, casein and ovalbumen.

25. A substrate according to Claim 23 or 24 wherein the components of said composition are provided at a concentration sufficient to saturate all of the binding sites on said substrate.

26. A substrate according to any of Claims 23-25 wherein the substrate is nitrocellulose.

27. A method to test whether an animal is intolerant to at least one food allergen comprising the steps of: i) contacting the body fluid sample in (i) with the composition according to any of Claims 1-22; ii) measuring or detecting the binding of antibodies in said body fluid sample with the allergens comprising the composition in (ii).

28. A method according to Claim 27 wherein the combined composition in (ii) is applied to the substrate according to any of Claims 23-26.

29. A method according to Claim 27 or 28 wherein said body fluid sample is selected from the group consisting of: blood or serum; semen; lymph fluid; cerebrospinal fluid; synovial fluid; tears; sweat; urine; saliva; or bone marrow.

30. A method according to Claim 29 wherein said body fluid sample fluid sample is blood or serum.

31. A method according to any of Claims 27-30 wherein the body fluid sample contains an immunoglobulin.

32. A method according to Claim 31 wherein said immunoglobulin is selected from the following Ig isotypes: IgA, IgM, IgD, IgE and IgG.

33. A method according to Claim 32 wherein said immunoglobulin is IgG.

34. A method according to Claim 33 wherein said IgG is selected from the group consisting of: IgGl, IgG2, IgG3 or IgG4.

35. A method according to Claim 34 wherein said body fluid sample is coupled or crosslinked or associated with a biotinylated food allergen to which is further added avidin-gold.

36. The use of a composition according to any of Claims 1-22 in an enzyme linked immunosorbent assay.

37. A kit comprising: an allergen composition according to any of Claims 1-24; a substrate according to any of Claims 25-27; detection means for the detection or measurement of allergen: antibody complexes; buffers and cofactors.