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Development of Test Systems for Allergy Testing: An Immunologist's Viewpoint

by Dr Michael Walker(more info)

listed in allergy testing, originally published in issue 28 - May 1998

An increasing number of people appear to be suffering from diseases which involve the immune system and sensitivity to different foods. Since the clinical problems occur some time after eating the trigger foods, we refer to these as hidden food sensitivities. In some patients this is reflected in an immune response to specific food proteins. Raised levels of food specific antibodies of the IgG, IgA and IgM classes can often be demonstrated in the serum of patients with food sensitivity, however, apparently normal individuals rarely have such antibodies.

Why patients produce antibodies to foods is not clearly understood at this time, however, it has been shown that if the reactive foods are removed from the diet, a significant number of patients see an improvement in their condition and general well-being.

Diagnostic methods for food sensitivity have been subjective and unreliable because of their poor reproducibility and accuracy. The most successful tests are those which test the immune reactions produced by different foods. To address some of these problems Dr Walker's team have developed an ELISA screening test which directly measures the activity of serum antibodies to a panel of 90 foods directly in a test plate. The test method is completely objective since it produces a photometric end-point.

Development of the food antibody assay system

The first stage is deciding which foods to include in the test panel and which to miss out. By consulting with nutritional experts we drew up a list of candidate foods which from experience were likely to cause sensitivity problems. These foods are shown in Table 1 below.

Table 1:
Ninety Food Antibody test

Grains
Rye
Barley
Buckwheat
Corn
Durum Wheat
Millet
Oat
Rice
Wheat


Dairy Foods
Cheddar
Cottage Cheese
Cow Milk
Egg
Goat MIlk
Yoghurt

Meats
Beef
Chicken
Lamb
pork
Turkey
Venison
Fish
Cod
Crab
herring
Lobster
Mackerel
Mussel
Plaice
Prawn
Salmon
Sole
Trout
Tuna

Vegetables
Aubergine
Avocado
Beetroot
Broccoli
Cabbage
Carrot
Cauliflower
Celery
Cucumber
Lettuce
Onion
Parsnip
Peas
Peppers
Potato
Spinach
Beans
Haricot Bean
Kidney Bean
Leek
Lentil
Soya Bean
String Bean

Fruits
Apple
Apricot
Banana
Blackcurrant
Grapes
Grapefruit
Kiwi Fruit
Lemon
Melon
Olive
Orange
Peach
Pineapple
Plum
Strawberry
Tomato
Nuts
Almond
Cashew
Coconut
Hazelnut
Peanut
Walnut

Miscellaneous
Chilli Pepper
Garlic
Pepper Black/White
Sesame Seed
Sunflower Seed
Tobacco Smoke
Chocolate
Coffee
Cola Nut
Mushroom
Cane Sugar
Tea
Yeast (Brewer's /
     Baker's Mix

Most foods are not suitable for use in the test in their raw form, and must be carefully processed by a variety of biochemical techniques to produce a relatively "pure" extract which contains the major proteins. The protein extract is then co-valently coated onto the surface of a polystyrene microtitre plate using proprietary technology developed at Genesis Diagnostics Ltd. Once fixed in position on the surface of the microtitre plate, the food extracts can be used in a food antibody test which relies on the very specific interaction between an antibody and its corresponding "food antigen". (See figure 1 below)

Showing the plastic surface of the microplate with food proteins immobilised onto the surface.

Figure 1 Showing the plastic surface of the microplate with food proteins immobilised onto the surface.

The coating process is repeated for all the required foods. Careful attention is given to the protein concentration of each food so that each one gives a similar response in the final assay enabling the concentration of one food antibody to be compared to another.

Once the food coated microplates have been prepared and stabilised, they are foil packed and will keep for up to 18 months.

The coated microplates are used in the following way.

The test involves three stages

Recognition and immobilisation of specific food antibodies
Removal of non specific antibodies using detergent solution
Visualisation and measurement of the concentration of specific food antibodies

1 Recognition of specific food antibodies


When a dilute solution of blood is allowed to come into contact with the plastic surface previously coated with food protein, any antibodies which recognise the food protein will become bound onto it like a key in a lock. (See figure 2 below)

 

Showing how different types of antibodies react with the food molecules.

Figure 2 Showing how different types of antibodies react with the food molecules.

2 Removal of non-specific antibodies

All other antibodies which do not recognise food do not bind and are washed away leaving only specific antibodies on the plastic surface. This is achieved by emptying the contents of the microplate and rinsing each well with buffer containing a detergent three to four times.

3 Visualisation of the antibodies

Any antibody which has bound to food molecules is retained after washing the microplate. To check if antibodies have bound to the plastic, an enzyme labelled antibody which is specific for human IgG is used. We use a goat-anti-human IgG antibody which has been chemically linked to a plant enzyme known as Horse-radish Peroxidase.

Various classes of antibody can be detected in this way, for example IgM, IgA and IgE. However, IgG is quantitatively the most important. IgG can be split further into four subclasses, and IgG subclass four has been implicated as important in some studies, however, the evidence is not conclusive and for this reason total IgG is preferred.

Each molecule of human antibody attached to the microplate is tagged by an anti-human IgG antibody-peroxidase molecule, as shown in figure 3 below. Any anti-human antibody which has not bound is washed away in a second series of rinsing stages using detergent solution.

Showing attachment of peroxidase labelled goat-anti-human antibodies to the patient's antibody.

Figure 3 Showing attachment of peroxidase labelled goat-anti-human antibodies to the patient's antibody.

The peroxidase molecule is an enzyme which has the property of transforming a clear solution into a coloured solution very quickly. This is the reaction that causes apples to go brown when they are damaged. The intensity of the colour produced is in direct proportion to the enzyme concentration and therefore to the original patient antibody concentration as shown in figure 4 below.

The enzyme converts a clear solution to intense yellow colour which absorbs light at 450 nanometers.

Figure 4 The enzyme converts a clear solution to intense yellow colour which absorbs light at 450 nanometers.

By comparing the colour intensity for each of the 90 foods to a set of calibrators, a concentration level can be calculated for each individual food. The colour density readings are performed in a microplate spectrophotometer as shown in figure 5 below.

Finger prick device/blood collection

Figure 5 Finger prick device/blood collection

Quality control and clinical studies

We have tested repeat samples to show that we get the same pattern of reactive foods every time the assay is run. The assay is very reproducible and it always gives the same pattern of food reactions when split patient samples are tested. Even different samples from the same patient taken several months apart will give similar results provided the patient's diet is stable.

Each time a new batch of test kits is made, a panel of patients with different food antibody patterns is used to assess the batch. In addition a control sample is included in the test kit so that laboratories who use our test kits can make a control check on each patient.

By testing large numbers of normal individuals, we have determined that values up to 12 U/ml of antibody is normal Values up to 25 U/ml are regarded as mild and above 50 U/ml as strong reactions.

It should be remembered, however, that the antibody concentration may not always reflect the severity of the problem. Some patients also have immune insufficiency diseases which may affect the results.

We have screened over 50 normal individuals without any apparent food sensitivity problems. In these apparently healthy individuals, the food antibody values were below 12 U/ml. In food sensitive individuals the immune reactions are very specific. For example most food reactions will be in the normal range except for isolated strong reactions to particular foods. This indicates that the patient has a specific anti-food reactions and the mechanism is unlikely to be due to simple leakage of food through the gut.

Often we see dramatically raised food IgG reactions in patients with suspected food sensitivity problems. For example, a raised level of milk antibodies of 100 U/ml or greater, often accompanied by lower levels for yoghurt and cheese and other milk related products. In other patients general grain reactions are observed with some or all grain antibodies raised. Sometimes only single grains such as wheat or oats react strongly. In other patients we have seen strong reactions to eggs, nuts, beans, olives, tomatoes or fish. The results can often be quite surprising.

The results can then be incorporated into planning an exclusion diet, avoiding completely all strongly reacting foods for a trial period of 3 – 6 months.

The food test is available world-wide from Immunology @ Home, a specialist service laboratory based in Cambridgeshire.

The food IgG test requires only a small amount of capillary blood. Samples can collected at home and sent by post to our laboratory. On ordering a test, a special blood collection kit is sent to the patient which allows blood to be collected at home.

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About Dr Michael Walker

Dr Michael Walker is an immunologist with over 25 years experience in immunoassay design and development. Dr Walker's Cambridgeshire based company, "Genesis Diagnostics Ltd" produces a wide range of immunological test kits which are supplied to Clinical Laboratories all over the world. Dr Walker became interested in the role of food antibodies about two years ago and has developed test kits for a variety of food antibodies. Contact Dr Michael Walker on 01353 669007 for further details.

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