Specific IgE

125I-labelled Bio-Line Allergo-Sorbent Test for the quantitative determination of specific IgE in human serum.

for in vitro diagnostic use only

Five different types of human antibodies have been characterized : IgA-IgD-IgE-IgM-IgG.In-vitro techniques for allergy testing have improved since the specific immunoglobulin responsible for allergic hypersensitivity was discovered and identified as IgE (1-2).Atopic allergy is a hypersensitive immunological condition mediated by IgE (3).Immunocompetent B-lymphocytes, if stimulated by a specific allergen, produce antibodies to the allergen.IgE antibodies bind, via their Fc portion, to receptors on the surface of most cells and basophilic leucocytes. Upon further stimulation, these cell-bound IgE molecules bind via their Fab portion to the allergen. This combination triggers cell degranulation and the release of various substances, including vasoactive amines.The most common clinical manifestations of this biological process are dermatitis, rhinitis, hay fever, asthma and anaphylactic shock.IgE determinations are most valuable in the diagnosis assessment of patients with established or suspected allergic diseases (4).IgE values are age-related.Some parasitic infections may also lead to increased IgE levels (5). Immunological studies of IgE myelomatosis have also been performed (6-8). The allergo-sorbent test on paper discs, developped in 1967 (9), is an in-vitro method for detecting IgE antibodies to specific allergens such as pollens, mites, house dusts, animal danders, foods, insect venoms, moulds and drugs.

The patient serum is first incubated in a test tube with a specific allergen covalently bound to a paper disc. Any antibody against this allergen will bind to the disc, if present in the serum.After the incubation period, the disc is washed, to remove unbound antibodies.In a second step, ¹²⁵I-anti-h-IgE is added, to fix and radiolabel the allergen-specific IgE antibodies.At the end of the test, the disc is washed again to remove the unbound tracer.The radioactivity is measured in a gamma counter and is directly proportional to the amount of IgE in the sample.The level of unknown IgE is then determined by comparing the radioactivity with data established using known standards in the same assay system.

Materials needed:

1. Reference set: 5 vials containing each 750 μl of ready-to-use sera for 5 duplicate curves, and 2 boxes containing each 25 reference discs (D1).

Standards: Zero (0.17 RU/ml), D (0.35 RU/ml),

C (0.7 RU/ml),  B (3.5 RU/ml),  A (17.5 RU/ml)

2. High reference serum H (optional): 1 vial of 750 μl (52.5 RU/ml).

3. ¹²⁵I-anti-IgE tracer: 1 vial (red solution) containing 5.2 ml of radiolabelled Mouse Monoclonal-anti-h-IgE. Activity per vial 6 μCi or 222 kBq. For 100 tests.

4. Control sera (optional): each vial containing 550 μl for 10 tests (3 types available).

5. Wash solution concentrate: 1 vial of 10.5 ml of concentrate, to be diluted into 1300 ml NaCl 9 ‰ and stored at 4° C (no more than 2 months).

6. Allergen discs: each box containing 25 specific or multiple allergen paper discs on a buffer substrate.

Sera should be separated from blood cells immediately after collection. Sera are stable for at least 7 days at 4^o C and for longer periods of time when stored frozen.

A) Assay procedure for the overnight technique in tubes:

Bring reagents to room temperature and mix before use. Label duplicate tubes for Zero,D,C,B,A (H optional) standards and Tc, and single tubes for control (optional) and patient samples. However, label 2 tubes per sample, when using specific allergen discs that must be used with HSA control discs.

1. Discs should be gently handled with tweezers and blotted on absorbent paper, to remove excess buffer solution. Place 1 reference disc into each standard tube, and 1 allergen disc into each sample tube. When allergen discs are provided with HSA control discs, place 1 allergen disc into one tube, and 1 HSA disc into a second tube.

2. Pipet 50 μl of standards, control and sample sera into their corresponding tubes (just above the disc), and slightly hit the disc with the pipette tip against the tube bottom, to eliminate any air bubble below the disc.

3. Cover with plastic film and incubate all the tubes for 3-4 hours at room temperature.

4. Aspirate, and wash all tubes, except Tc, three times with 2 ml of wash solution, with 10 minutes incubation between each wash. Aspirate and discard as much remaining liquid as possible.

5. Pipet 50 μl of tracer into each tube, including Tc, using the same procedure as in step 2.

6. Cover with plastic film and incubate all the tubes overnight (16-24 hours) at room temperature.

7. Repeat wash procedure as described in step 4.

8. Place tubes in gamma counter and count for one minute.

Data need not be expressed as counts per minute (cpm) but the counting period must be the same for all tubes that are counted.

Determine the average counts for each set of duplicate tubes. Divide this value by the average net counts of Tc, and multiply by 100 to yield the % B/Tc.

% B/Tc = cpm (Stds, Controls or sample) x 100/cpm (Tc)

Plot % B/Tc for each standard vs its concentration in RU/ml. The concentration of Specific IgE in the samples may then be read directly from the standard curve.

No shorter technique is recommended; however, possible alternatives between both techniques can be implemented by each laboratory, according to their needs. Our quality control is based on the overnight technique.

1. Specific Allergen Discs.

Results may be expressed either way: in classes (0, 1, 2..), or in RU/ml (Rast units/ml): 0 RU/ml < class 0 < 0.35 < class 1 < 0.7 < class 2 < 3.5 < class 3 < 17.5 < class 4 < 52.5 < class 5.

In each case, the counts/min (cpm) of the samples have to be compared with the cpm of the reference standard curve.

Expected values: class 0: negative

class 1: suspected

class 2: low

class 3: high

class 4: very high

2. Multiple Allergen Discs.

Values equal to or higher than class 1 are considered as positive; they indicate that specific IgE antibodies to one or more allergens are present in the serum.

3. Allergen Discs with Control discs.

Some allergen discs are delivered with HSA (Human Serum Albumin) control discs. Due to the coupling of those allergens with HSA on discs, results are considered as positive when

the cpm of the sample disc are higher than twice those of the HSA control disc.

1. Ishizaka K T, Hornbrook MM. Physico-chemical properties of human reagenic antibody. IV. Presence of a unique immunoglobulin as a carrier of reagenic activity.J Immunol 97, 75-85 (1966).

2. Johansson SGO, Bennich H. Immunological studies of an atypical (myeloma) immunoglobulin.Immunology 13:381-394 (1967).

3. Frick OL. Immediate hypersensitivity. In Basic and Clinical Immunology, HH Fundenberg, DP Stites, JL Caldwell, JV Wells, Eds., Lange Medical Publications, Canada, 1976,pp204-224.

4. Johnsson, S., Bennich, H., and Berg, T., Progress in Clin. Immunology, 1.(1972).

5. Gleich, G., Averbeck, A., and Swedlund, H., J. Lab. and Clin.Med. 77:690 (1971)

6. Koh T,Ohno T, Kageyama S, et al. IgE multiple myeloma: a case report and review of the literature. Acta Haematol Jpn 49:696-702 (1986).

7. Hegewisch S, Mainzer K, Brauman D.IgE myelomatosis; presentation of a new case and summary of literature. Blut 55:55-60 (1987).

8. Van Wijk HJJ, Kerckhaert JA, Oei OL, Van Helden HPT. IgE myeloma: case report and review of the literature. Neth. J. Med. 29:196-200 (1986).

9. Wide L., Bennich H. and Johansson SGO. Diagnosis of allergy by an in-vitro test for allergen antibodies. Lancet, V.2:1105 (1967).