Total IgE liquid Phase

Radiometric assay kit for the quantitative determination of total serum IgE.

BL-05-100

for in vitro diagnostic use only

Summary and background of the test:

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).

Various techniques provide accurate and reliable results: immunoradiometric assay, chemiluminescent immunoassay (9-10), nephelometric immunoassay (11), biotin-avidin based solid-phase (12) and radial partition fluorometric enzyme immunoassay (13).

Principle of the test :

The Bio-Line Total IgE liquid phase IRMA kit is a two-site immunoradiometric assay for the quantitative determination of IgE.

A first Mouse monoclonal antibody is immobilized onto the plastic tube. A second Goat polyclonal antibody is radiolabelled.

The sample is incubated with the solid-phase antibody-coated tube. After the incubation period, the tube is decanted and washed. Only the antigen from the patient sample remains bound onto the tube.

In a second step, the radiolabelled ¹²⁵I-antibody is added. If IgE is present in the sample, a "sandwich" complex is formed :

 2^nD Ab.>--<Rabbitl anti-h-IgE>--<sample IgE>--<¹²⁵I Goat anti-h-IgE.

At the end of the test, the tubes are washed to remove unbound tracer and other unbound components. 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 IgE standards in the same assay system.

Precautions:

1. Radioactive material: Radioactive material may be received, acquired, possessed and used only by physicians, clinical laboratories, or hospitals for "In-Vitro" clinical or laboratory tests not involving internal or external administration of the material, or the radiation therefrom, to human beings or animals.

Compliance with these basic rules of radiation safety should provide adequate protection:1. Do not eat, drink, smoke, or apply cosmetics in areas where radioactive material is used.

                2. Do not pipet by mouth reagents containing radioactive materials.

                3. Wear protective clothing; i.e., lab coats and disposable gloves, in order to avoid direct contact with radioactive reagents.

                4. Work with radioactive materials should be performed in a designed area.

                5. Radioactive materials should be stored in an acceptable location.

                6. A log should be kept for receipt and disposal of radioactive materials.

                7. Radioactive spills or accidents should be taken care of immediately according to established procedures.

                8. Disposal of radioactive materials must comply with prevailing regulations and guidelines of the agencies holding jurisdiction over the laboratory.

2. Sodium azide: Sodium Azide, used as a bacteriostatic agent, is toxic in acid medium. In addition, it may form potentially explosive lead or copper azides. To avoid dangerous deposits, waste solutions should be flushed away with large volumes of water.

3. Hepatitis and Acquired Immune Deficiency Syndrome (HTLV-III): All Bio-Line reagents included in this kit have been tested and found to be non reactive for hepatitis B surface antigen. They have also been screened and determined to be non-reactive for HTLV-III antibody. However, human serum products should be handled as if potentially capable of transmitting hepatitis, Acquired Immune Deficiency Syndrome, or other infectious agents.

Materials provided

Kit contains sufficient reagents for 100 determinations.

1.             Total IgE standards & control: 10 vials containing each 500 μl except the Zero 2 ml.

                Standards: 0-2-5-20-50-200-500-1000-2500 IU/ml                 & control: 130 ± 30 IU/ml. Standards and control have                 been calibrated against the IRP 75/502.

2.                    ¹²⁵I-anti-IgE tracer: 1 vial (yellow solution) containing 21 ml of radiolabelled Goat-anti-h-IgE + Rabbit Polyclonal. Activity per vial £ 12μCi or £ 440 kBq. Ready for use.

3.             Second Antibody: 1 vial of 26 ml of Sheep anti Rabbit Ab.stored at 4°C.

Reagents provided should be stored at 2^o - 8^o C.

Refer to the expiration date on the kit label for stability.

Materials required but not provided:

1.             Pipets, micropipets, repeating syringes and repeating pipettors.

2.             9 ‰ NaCl saline solution.

3.             Gamma counter.

4.             Logit log graph paper.

5.             Test tube racks.

6.             Vortex mixer.

7.             Water bath at 37°C.

Specimen collection and preparation:

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.

Assay procedure :

Bring reagents to room temperature and mix before use. Label tubes for total counts (Tc), standards, control sera and unknowns.

1.                    Pipet 50 μl of standards, control and samples into tubes just above the assay buffer.

2.                    Pipet 200 µl of tracer.

3.                    Vortex all the tubes or shake the entire rack with a side to side motion.

4.                    Cover with plastic film and incubate all the tubes in water bath for 1 hour at 37°C.

5.                    Pipet 250 μl of  2nd Ab. into each tube, except Tc 

6.                    Mix and Incubate RT 15 minute.

7.                    Add 2 mL of NaCl 0.9% before centrifugation step.

8.                    Centrifuge at 4°C during 20 minutes

9.                    Decant or aspire.

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

Data table (Example).

Calculation of results:

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 IU/ml. The concentra­tion of Total IgE in the unknown samples may be read directly from the standard curve.

Samples exhibiting titers greater than 2500 IU/ml should be diluted using the standard Zero as diluent. If necessary, multiply by the dilution factor to determine the initial concentration.

Expected Values:

The values below are indicative. Each laboratory should establish its own normal range. Pediatric values are available on request.

Lower than 20 IU/ml:        allergy not probable.

Between 20 and 100 IU/ml: allergy questionable.

Higher than 100 IU/ml:        allergy very probable.

Bibliography:

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 1986;49:696-702.

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

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

9. Christopher R. Brown, Keith W.Higgins, Kelly Frazer, Linda K. Schoelz, John W. Dyminski, Vincent A. Marinkovich, Steven P.Miller, and John F. Burd. Simultaneous Determination of Total IgE and Allergen-Specific IgE in Serum by the MAST Chemiluminescent Assay System. Clin. Chem. 31/9, 1500-1505 (1985).

10. JP Basuyau, Ph Brunelle, M. Leroy and MP Vigier.Chemiluminescent Assay For Total Serum IgE. Clin.Chem., Vol. 35, N^o6, Pg 1194 (1989)

11. Wolfgang H. Kapmeyer. Automated nephelometric immunoassay of immunoglobuline with novel shell/core particles. Clin.Chem. , Vol 35, N^o6, 1989.

12. Hann-Ping Wang, Molly Daniel, Barbara Williams, and William Knight (Beckman Instruments, Brea, CA 92621). A BIOTIN-AVIDIN based solid-phase enzyme-linked immunosorbent assay for human serum IgE. Clin.Chem. ,Vol. 35, N^o6,Pg 1192.(1989)

13. Niranjan Patel, M. Oppermann and C. Bowman.Evaluation of radial partition immunoassay for the quantitative determination of immunoglobulin E (IgE). Clin.Chem., Vol. 35, N^o6,Pg 1199,(1989).