The following points highlight the four main types of diagnoses of allergy. The types are: 1. Case History 2. Skin Testing 3. Provocation Test 4. Diagnosis of Allergy based on Total and Specific IgE Determinations.
Contents
Type # 1. Case History:
The case history holds a central position in allergy investigations. In order to get a comprehensive idea about the source and nature of allergens the physician will question the patient or ask him/her to fill in a questionnaire.
It is very important to know the time and nature of symptoms that develop, whether it has any relationship with the seasons, weather, physical activity etc. Some other information are also very important like a knowledge of personal habits such as smoking, occupation, hobbies, etc.
The case history alone may give enough information to settle the diagnosis. However, further investigations have to be performed to get a final diagnosis.
Type # 2. Skin Testing:
Skin testing is the primary tool of allergy diagnosis. This can be done either as a prick test or scratch test or as an intracutaneous test. The scratch testing has now largely been abandoned due to lack of precision.
i. Prick Test:
Tests are performed with the suspected allergen solution placed on the ventral side of the forearm and each site is pricked with a disposable hypodermic needle (Fig.10.11). The wheal responses are measured after 15-20 minutes and grades 1+ to 4+ according to the suggestion of Stytis (1982) (Table 10.6). This may identify and confirm the allergy.
ii. Intradermal (Intracutaneous) Test:
An equeous extract of the suspected allergen is injected superficially into the skin, giving a 3 mm bleb. The extract used are 1000 – 10,000 times diluted than those used for prick testing. This test is 10 -100 times more sensitive than prick testing.
Intradermal testing is generally not performed, because it involves the risk of anaphylactic shock and is painful for the patient. It is rarely performed in those cases where prick testing gives negative or doubtful results.
Positive and negative controls:
A negative control with acid phosphate (100 mg/ml) and buffered saline diluent is employed. A positive control with histamine diphosphate (1 mg/ml for prick test and 0.01 mg/ml for intradermal tests) is used to judge the reactivity of the skin.
Advantages of prick test:
Prick testing is recommended widely both for children and adults. It is highly specific and involves only minimal risks of anaphylactic reactions. It is easy to standardize and to perform and gives little pain to the patient.
Factors influencing skin reactivity:
Antihistamine depresses skin reactivity considerably, hence antihistamine treatment must be discontinued someday (~ 4 days) before testing.
Type # 3. Provocation Test:
Sometimes provocation test is performed in the eyes and nose of hay fever patients and in the airways of asthmatic patients.
For hay fever patients, a highly diluted allergen extract is dropped into the nose and eyes or is inhaled. The test may be continued in this way using more concentrated allergens until the allergic symptoms are provoked. In this test, the tested allergens which are involved in patients symptom can be identified.
The provocation test, apart from being inconvenient to the patient, has other disadvantages. It has risk and may be influenced by physical and mental condition of the patient.
Type # 4. Diagnosis of Allergy based on Total and Specific IgE Determinations:
The skin and provocation tests have many limitations and disadvantages, hence there is a need for more convenient and reliable methods. The IgE plays an important role in allergy. The healthy non-atopic persons have a very low level of IgE (14 unit/ml) in serum, while in atopic patients significantly increased levels of IgE (>100 unit/ml) are found.
Therefore, the measurement of IgE level is of great importance when diagnosing allergies. There are various immunological tests which accurately measure IgE, even a small blood sample is sufficient for allergy testing.
The measurement of total IgE (by PRIST, ELISA, etc.) can indicate the symptoms of allergic origin and the measurement of specific IgE (by RAST) can predict the specific allergen responsible for such allergy. Because of their ease of use, reliability, convenience and accuracy, these tests form an important part of the diagnostic work.
These techniques are necessary and are commonly used in the following situations:
(a) Where skin-testing is not possible. Viz.
i) Among children below five years of age.
ii) For mentally retarded persons.
iii) Among patients who are in treatment of antihistamine.
iv) Among patients who are suffering from chronic dermatitis.
(b) Skin-testing sometimes may produces doubtful results.
(c) To arrive a definite conclusion after getting positive test results of skin-test, other allergological diagnosis are;
i. Radio Immuno Assay (RIA):
RIA is an exceptionally powerful device in diagnosis of allergy. Rosalyn Yalow awarded the 1977 Nobel Prize in Medicine for development of RIA. In this method a purified radio-isotope- labelled antigen or antibody is used which competes for antibody with unlabelled standard or antigen in experimental samples.
Then, the radioactivity of antigen or antibody is measured by radioisotope analysers. Several sensitive RIA methods have been developed such as RAST (Radio Allergo Sorbent Test), RIST (Radio Immuno Sorbent Test), PRIST (Paper Radio Immuno Sorbent Test), and the CAP-System ECP-Immunoassay for detection of IgE-levels in suspected atopic patients.
a. RAST:
It is a laboratory investigation for the determination of specific IgE antibody. RAST is a two-step immune reaction. In the first step, the antigen is bound to the solid phase, then incubated with patients sera. So that IgE molecules specific to the antigen bind. In the second phase, labelled anti-IgE serum is added, which combines with attached IgE. The radioactivity of the solid phase is measured to identify the serum level of specific IgE antibody.
The RAST is semi-quantitative and the results are graded in a scale from 0-4. Here, serum analysis will not identify the organ to which the allergic symptoms are localized. RAST result provides rather poor information on the degree of clinical hypersensitivity. Hence RAST results should be evaluated together with the case history and the results of other techniques for allergy diagnosis.
b. PRIST:
The measurement of total serum IgE in patients is found to be a sign of atopic status and allergy. As the serum IgE level is very low, sensitive immunoassays like PRIST, CAP System ECP-immunoassay, etc. are required.
The principles of total IgE measurement by PRIST have been given below:
c. ECP – Immunoassay:
In atopic allergic disease, the eosinophils and the mast cells are the most notable cells. They become activated, degranulated and release mediators and proteins, hence measurement of these molecules seems to be a rational way of making a diagnosis of allergy.
Eosinophil cationic protein (ECP)-immunoassay in the Pharmacia CAP System is a modern tool to measure the total serum IgE in patients. The measurement of radioactivity is done in Gamma Counter in case of 125I labeled anti – ECP, which can be replaced by a fluorescent agent (FEIA). The summery of the ECP – immunoassay has been given in Fig.10.12.
The major disadvantages of different radio-immunoassays include:
(a) The relatively high cost of equipment (e.g., Gamma Scintillation Counter) and reagents (e.g., radioiodine),
(b) The radiological hazards of using radioisotopes,
(c) The short half-life of reagents (the half- lives of 12SI and 131I are 60 days and 8 days respectively),
(d) Assays usually take place for days rather than hours.
ii. ELISA:
ELISA (Enzyme-Linked Immuno Sorbant Assayjcombines the activity of antibodies or antigen or hapten conjugates to an easily assayed enzyme which also possesses a high turnover number.
ELISA replaces radioimmunoassay techniques like RAST, PRIST, etc., because:
1. ELISA is more sensitive than RAST and PRIST.
2. It is relatively cheap to operate.
3. It lacks the radiological hazards of radioimmunoassay.
4. Not much costly instruments, chemicals and space are required, so it is suitable for use in laboratories of the developing countries.
5. A very small quantity of allergen or antibody can be detected.
6. Assays usually take short time (hours) than the time required (days) for RAST, PRIST, etc.
In stead of radioactive elements, enzymes are used in ELISA. So it is very important to select the appropriate enzyme.
The enzymes should fulfill the following criteria:
i) The properties of enzyme should remain unchanged during its storage or assay.
ii) The activity of the enzyme should remain fixed.
iii) The enzyme should be easily available.
iv) Blood serum of the patient should be devoid of such enzyme.
At present, nine different types of enzymes are detected and isolated from higher plants, animals and E. coli bacterium.
They include:
1. Horse – Raddish peroxidase
2. Alkaline phosphatase
3. Glucose oxidase
4. Glucoamylase
5. Egg-white Lysozyme
6. Malate dehydrogenase
7. β – Glucosidase
8. Glucose 6 – phosphate dehydrogenase
9. Acetyl choline esterase
Generally for the detection of IgE in atopic patients, Alkaline Phosphatase or Horse – Raddish Peroxidase is used.
Solid phases used in ELISA include crosslinked dextran or polyacrylamide beads, filter paper (cellulose) discs or polypropylene tubes. Disposable polystyrene microtitration plates are generally convenient for large numbers of samples.
Antigen or antibody is attached to the solid phase by passive adsorption or covalent coupling with cyanogen bromide. Test samples of antigen or antibody are usually diluted with the same buffer containing a wetting agent used for washing the antigen-bound solid phase.
The antibody- enzyme conjugate used must contain a high reactive antibody coupled to an enzyme with a high turnover number. Enzyme substrates should ideally be stable, safe and inexpensive. Reference positive and negative samples must be included in each series of tests to ensure accurate and reproducible results.
ELISA is used for assaying antigens either by a competitive method or by a double antibody sandwich method or by indirect method. All these methods require the preparation of a calibration curve during the assay.
a. Competitive method:
In this method a mixture of a known amount of enzyme-labelled antigen and an unknown amount of unbound antigen is allowed to react with a specific antibody attached to a solid phase. After washing with buffer, the enzyme substrate is added and the enzyme activity is measured spectrophotometrically.
The major disadvantage of this method is enzyme-labelled antibody. After further that each antigen may require a different method to couple it to the enzyme.
b. Double – antibody sandwich method:
In this method the unknown antigen solution is reacted with specific antibody attached to a solid phase, then washed and treated with washing the enzyme substrate is added and the enzyme activity is measured spectophoto- metrically.
The amount of measured enzyme activity is directly proportional to the amount of an antigen present. An advantage of this method is that only one procedure is required to couple the enzyme to all antibody preparation.
c. Indirect ELISA method:
This method is used for the detection and measurement of antibody. The test antisera is reacted with specific antigen attached to a solid phase. Any specific antibody molecules can bind to the antigen.
Enzyme labelled antihuman antibodies are added which will bind to any specific antibody molecules adsorbed from the original serum. The complex is washed and the Summary of indirect ELISA method: enzyme substrate is added resulting in activity proportional to the amount of specific antibody in the original serum.
iii. Western Blotting:
In a mixture of proteins or fragments of proteins Western blotting technique is used to detect those that react with the same antibody. In this technique the antibody-reactive allergens (proteins) are analysed by one-dimensional denaturing gel electrophoresis. Then the gel is placed in contact with a sheet of nitrocellulose, and the proteins are transferred (or ‘blotted’) to the nitrocellulose.
The proteins are bound irreversibly to the nitrocellulose sheet, so the antigen-antibody reactions can be visualized after treatment of the sheet with radio-labelled antibody or a form of the ELISA technique or by chemiluminescent procedure.
