Add as bookmark

Allergy Testing in the Laboratory

by Mark Varey(more info)

listed in allergy testing, originally published in issue 15 - October 1996

Adverse reactions to foods have been recognised for thousands of years; "what is food to one man may be fierce poison to another" Lucretius, circa 75 BC.

The term allergy is derived from two Greek words which mean "altered reactivity". An allergy is an immune reaction to a normally harmless substance, be it food or environmental agent such as dusts, pollens or chemicals. Allergic reactions are termed hypersensitivities.

Before discussing available laboratory tests for hypersen- sitivities it is important to understand the basics of the immune system[1]. The immune system is the body's protection against invasion by harmful micro-organisms and other foreign matter. Substances which act as triggers to the immune system are called antigens. The immune system responds to the presence of antigens in two ways:

a) producing antibodies (humoral response)
b) producing large numbers of lymphocytes (cell mediated response).

An important property of the immune system is that it is specific to the antigen which triggered it, it will not react to other harmless molecules even if they are of similar structure. The immune system has a "memory", if it has reacted once to a particular antigen, it will react rapidly and vigorously the next time it is presented with the same antigen.

Foods may affect the individual by a number of mechanisms, some involving the immune system and some not. The various immune mechanisms involved in the production of tissue damage have been classified, by Gell and Coombs2, into four distinct types, three types are antibody mediated and one is cell mediated.

Type I: Anaphylactic or Immediate Hypersensitivity – is the classic allergic type of reaction mediated through the antibody of the IgE class. White blood cells called mast cells and basophils have IgE sticking to their outer surfaces. When the IgE combines with a foreign antigen, a mechanism is activated which causes the mast cell or basophil to release very potent chemicals such as histamine. These chemicals cause tissue damage and inflammation resulting in often dramatic symptoms, including allergic asthma, acute gastrointestinal distress, urticaria and anaphylactic shock, such reactions are potentially life threatening.

Type II: Cytolytic Reactions – cytolytic (cell splitting) hypersensitivity reactions involve the binding of IgG and IgM antibody to foreign antigen. Antigen-antibody binding results in activation of the complement cascade (an enzymic system in the blood), causing destruction of the antigen and damage to tissues.

Type III: Immune complex hypersensitivity – circulating immune complexes are formed when antibodies bind to soluble antigen. Immune complexes are cleared from the system by white blood cells called macrophages. However, when there is an excessive build up of immune complexes, the macrophages are unable to clear them up quickly enough. Excess immune complexes are deposited in tissues causing injury and inflammation.

Type IV: Cell-mediated hypersensitivity – is the result of reactions in the tissues between T-lymphocytes (white blood cells) and antigens, causing the release of chemical factors giving rise to inflammation. Cell-mediated hypersensitivity occurs without the involvement of antibody or complement. Contact dermatitis is an example of a type IV reaction.

Symptoms of type I hypersensitivity occur immediately, usually within an hour of eating the offending food. The reactions are violent and it is often obvious which food is the culprit. Certain food allergens are commonly associated with immediate reactions and include nuts, fish, shellfish, eggs, legumes and strawberries. Once sensitised the individual always remains allergic to the food. Fortunately these 'true allergies' affect only a small percentage of the population.

The delayed food reaction is most probably a combination of types II and III hypersensitivity[3] . Symptoms usually are triggered by food ingested daily. The symptoms tend to be chronic or recurrent, occurring hours to days after ingestion of the food, and persist for days after the food is omitted. The relationship between the food and the reaction is rarely suspected. The more often the food is ingested, the less it is tolerated.

Avoidance of the food for an appropriate interval, usually 3 to 6 months, increases tolerance to the food when next exposed. Delayed food reactions are more common than true immediate food allergy and are usually termed 'sensitivities'. Chronic, recurrent symptoms are inflammatory in nature and associated with multiple organ system involvement. The symptoms induced by the delayed reactions are diverse and include:

Respiratory – rhinitis, sinusitis, asthma and otitis media.
Gastrointestinal – vomiting, bloating, cramping, gas, distension, nausea, diarrhoea and colic.

Dermal – urticaria, atopic dermatitis, eczema and other rashes.

Central Nervous System – migraine, headache, impaired concentration, altered alertness, mood and behavioural changes, depression and hyperactivity.

Musculoskeletal – arthritis, aching, weakness, myalgia and cramping.

Interestingly, it has recently been reported4 that early loosening of total hip replacement may, in some cases, be due to an allergy to a constituent of bone cement used in the operation.

Available Laboratory Tests

There are basically two classes of allergy testing. Tests to identify type I reactions, and tests to identify delayed types II and III reactions.

Type I – there are several methods to determine antigen-specific IgE in blood serum, the two main techniques are radioimmunoassay (e.g. RAST) and enzyme-linked immunosorbent assay, though other methods based on chemiluminescence or fluoroimmunoassay are available. The RAST (radioallergosorbent test) is probably the most widely used.

The principle of the RAST test is straight forward5. A cellulose paper disc, or other suitable solid-phase support, labelled with antigen which might be a food, inhalant, venom or other antigen, is incubated with a small amount of the patients blood serum. During incubation antigen-specific IgE, if present, binds with the antigen.

The disc is washed to remove any unbound IgE. Anti-human IgE, tagged with a radioactive label, is added and allowed to incubate long enough to combine with the antigen-specific IgE. After further washing to remove unbound, radio labelled anti-IgE, the level of radioactivity of the disc is measured. The level of radioactivity is directly proportional to the amount of antigen-specific IgE.

RAST has the advantage of being very sensitive, probably more so than skin tests, the tests are performed in vitro, therefore pose no risk to the patient. Skin testing may potentially provoke an anaphylactic reaction in patients with an exceptionally high level of sensitisation. The RAST test is useful in diagnosing allergies to dust, dust mite, animal danders, moulds, pollens, venoms and some foods.

The disadvantage of RAST is it only identifies immediate reactions, most food related reactions are delayed.

Other classes of antibodies with the same antigen specificity, particularly IgG, can interfere with IgE binding and produce a false-negative result.

Some atopic patients have an extremely high level of total IgE, this can cause a false positive result through non-specific binding of IgE on the test material.

Type II and III – there are two techniques available to determine delayed reactions to foods. The cytotoxic test6 assesses the degree of reaction of live white blood cells when challenged by antigen. More recently, the measurement of food specific immunoglobulin G (IgG),7,8,9,10 has been investigated for the identification of food sensitivities.

The cytotoxic test involves centrifuging a sample (10 ml) of the patients blood to allow removal of the white blood cells and plasma. A suspension of white blood cells and plasma is placed in contact with each of the pure food extracts to be tested, in a microscope chamber. After a period of incubation the result is obtained by assessing the degree of reaction of the cells.

The principle cells involved are the neutrophils, these are phagocytes which engulf 'foreign' material and destroy it. The degree of reaction is assessed using a microscope to observe the number of vacuoles or bubbles formed inside the neutrophils. These are the results of phagocytic activity; the greater the number of vacuoles, the more vigorously the cell has been attacking foreign, 'invading' protein. In extreme reactions this may lead to the neutrophil itself dying and bursting, to leave a 'ghost cell'.

Cytotoxic testing has several advantages, it is in vitro. It is very sensitive, being capable of detecting mild sensitivities. Many foods can be tested using a single blood specimen. It is economical.

The disadvantages are it is subjective, being around 75% reproducible. Results may be affected by drug therapy. Live blood cells are required so blood specimens are very time sensitive. Patients are required to fast for 12 hours before having blood drawn.

The test for food specific IgG is performed using enzyme linked immunosorbent assay (ELISA), to detect circulating antibodies, which recognise and bind to purified food antigens. In the ELISA method, food antigens are immobilised on to the surface of 96 well polystyrene micro-plates. The patients diluted blood serum is added to the micro-plate wells and allowed to incubate with the food antigens for a fixed time.

If antibodies specific to a certain food are present, they will attach themselves to the food antigen. The micro-plates are then rinsed to remove other unattached antibodies and blood constituents. The food specific antibodies are then detected using a second antibody (anti-human IgG), which is chemically tagged with an enzyme. The second antibody binds with the food specific antibodies.

When a substrate is added the enzyme 'tag' causes a colour to develop. The colour density obtained is an accurate indication of the original anti-food antibody concentration in the patients blood specimen. The colour produced by calibrated reference standards is compared with the patient's result, allowing the laboratory to accurately estimate the amount of each food antibody. A positive control sample should always be included in each test to confirm that the assay is working correctly.

The test has many advantages, it is in vitro. It is accurate and reproducible, greater than 95% agreement between split specimens is possible. It measures all four IgG subsets. The test requires low blood volumes, 90 foods can be tested using 0.1 ml of serum. Blood specimens are relatively time stable. Patients are not required to fast before having blood drawn.

The disadvantage is it is relatively expensive when compared to cytotoxic testing.

How To Use The Test Results

With regard to type I hypersensitivity, the only course of action is to strictly avoid the offending substance. Fortunately in the case of foods, reactions are usually limited to a single food antigen or food family such as nuts or white fish. Often only the slightest trace of antigen is enough to trigger a severe reaction, so great care must be taken to avoid 'hidden' ingredients, particularly in processed foods.

In the case of type II and III hypersensitivities, a number of foods are often involved. The patient should be advised to avoid those foods showing the higher degree of reaction, and to reduce their intake of foods showing a moderate degree of reaction. Often the moderate reactors can be eaten on a four day rotation diet, i.e. each food should be eaten no more frequently than once every four days.

When foods are first eliminated from the diet the patient may experience withdrawal symptoms, that is symptoms (e.g. headache, eczema or joint pains) may become worse in the first few days. Perseverance is required as improvement may only become apparent anything from a few days to a few weeks into the diet.

Patients are normally advised to comply with the diet for at least 3 months, after which time the reactive foods can be reintroduced to determine which foods continue to cause problems. This should be done in a controlled manner, reintroducing one food at a time and leaving at least a week between each food. In this way it is easy to determine which food is responsible for any return of symptoms.

Test results must be interpreted in conjunction with other clinical information relating to the patient. In particular the underlying cause of food sensitivities should be investigated to prevent the development of new sensitivities. One reason for the development of food sensitivities might be increased intestinal permeability or 'leaky gut syndrome'.

Intestinal permeability allows relatively large molecules, including incompletely digested foods, to pass through the intestine into the blood stream where they trigger the immune response and cause inflammatory symptoms.

Intestinal permeability has been reported in several conditions including:

* Food sensitivities
* Intestinal candidiasis
* Coeliac disease
* Alcoholism
* Chron's disease

Other factors which increase the likelihood of developing food sensitivities include:

* Eating certain foods too frequently
* Eating too much at one time
* Eating too rapidly and under-chewing
* Eating deep fried foods
* Eating over cooked foods
* Eating over processed foods (canned foods, refined flour, refined sugar, etc)
* Eating while emotionally upset
* Eating food while drinking alcohol

There are many non-immunological reactions to foods[11] which can not be identified by the laboratory techniques described, these include:

Natural Pharmacologic Agents – pharmacologically active substances present in a variety of foods can cause adverse symptoms. Vasoactive amines such as tyramine, serotonin and histamine are found in cheeses, chocolate, fish, bananas, red wine, citrus fruits, strawberries and other foods.

Naturally Occurring Toxins – many foods contain small quantities of toxic agents, which can cause problems if eaten in large quantities. Examples of such foods are kidney beans, nutmeg, wild mushrooms, almonds, rhubarb, tea, peppers and many others.

Metabolic Reactions – metabolic reactions to foods are caused by the ingestion of usually safe foods by persons who are susceptible because of disease states, inborn metabolic problems or medications. Such conditions include milk intolerance due to lactase deficiency and diabetes due to insulin deficiency.

Reactions to Food Additives – thousands of additives are available for use in processed foods. These include emulsifiers, stabilisers, preservatives, colourings, flavourings etc. In susceptible people they can produce a range of symptoms including headache, nausea, diarrhoea, urticaria and asthma. Non-immunological reactions might be investigated using dietary elimination and subsequent challenge techniques.


It can be seen that foods may produce adverse effects in susceptible individuals through a variety of immunological and non-immunological mechanisms. There are laboratory techniques available to identify immunological reactions. The appropriate technique should be selected on the basis of the individual's clinical history.


1. Rowley, N. Basic Clinical Science. Hodder and Stoughton, London. 1994.
2. Coombs, R.R.A. & Gell, P.G.H. Classification of allergic reactions responsible for clinical hypersensitivity and disease. In P.G.H. Gell, R.R.A. Coombs & P.J. Lachmann (eds.), Clinical Aspects of Immunology (3e), p. 761. 1975.
3. Trevino, R.J. Immunological Mechanisms in the Production of Food Sensitivities. The Laryngoscope 91. p. 913-1936. 1981.
4. Haddad, F.S. et al. Hypersensitivity in Aseptic Loosening of Total Hip Replacements. J Bone Joint Surg [Br]. 78-B. p. 546-9. 1996.
5. Gooi, H.C. & Chapel, H. Clinical Immunology, A Practical Approach. Oxford University Press. New York. 1990.
6. Bryan, W.T.K. and Bryan M.P. The Application of In Vitro Cytotoxic Reactions to Clinical Diagnosis of Food Allergy. Laryngoscope LXX, 6. p. 810-824. 1960.
7. Kemeny, D.M. et al. Sub-Class of IgG in allergic disease. IgG Sub-Class Antibodies in Immediate and Non Immediate Food Allergy. Clinical Allergy. 16. p. 571-581.1986.
8. Rafei, A. et al. Food Allergy and Food Specific IgG Measurements. Annals of Allergy. 62. p. 94-99. 1989.
9. Halpern, G.M. et al. Annals of Allergy. 58. p. 14-27. 1987.
10. Bock, S.A. et al. J Allergy Clin Immunol. p. 165-174. 1986.
11. Fadal, R.G. Introduction to Food Allergy and Other Adverse Reactions to Foods. Resident & Staff Physician. June, 1988.


  1. No Article Comments available

Post Your Comments:

About Mark Varey

Mark Varey, M.I.Sc.T

  • SensiStop!

    All Natural Medicines for Allergy Asthma Eczema Candida Periodontitis, a safe allergy treatment.

  • The Dove Clinic

    Treatment of: IBS; Allergy; Angina; CFS; Life Threatening Illness; consultations in chronic illness.


    I use many therapies to help my patients: acupuncture, vega testing, naturopathy, herbal medicine.

top of the page