Total IgE ELISA - pNPP 

Direct enzyme-linked immunosorbent assay kit for the

quantitat­ive determination of total serum IgE.

BL-E-05-96

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).

The radioactive label is still widely used in IgE determinations. Other  techniques provide also adequate and reliable results: chemiluminescent immunoassays (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 microplates Elisa kit is a two-site enzyme-linked immunosorbent assay for the quantitative determination of IgE.

A first Mouse monoclonal antibody is immobilized onto the plastic wells.

A second polyclonal antibody is labelled with the alkaline phosphatase enzyme.

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

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

Well---Mouse Monoclonal anti-h-IgE---sample IgE---conjugate polyclonal anti-h-IgE.

At the end of the second incubation, the wells are washed to remove unbound conjugate and other unbound components.

A third incubation is performed with the chromogen (pNPP).

The reaction is then stopped with NaOH, and the plate is read at 405 nm in a spectrophotometer.

The color intensity is directly proportional to the amount of IgE in the sample. The level of unknown IgE is then determined by comparing the optical density with data established using known IgE standards in the same assay system.

Precautions:

1.             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.

2.             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 96 determinations.

1.             Total IgE standards & control: 7 vials containing each 500 μl, except Zero 1 ml. Control: 150 ± 50 IU/ml. Standards: 0-2-5-20-50-200-500 IU/ml, calibrated against the IRP 75/502.

2.             Anti-IgE alkaline phosphatase conjugate: 1 vial containing 10.5 ml of ready-to-use polyclonal anti-h-IgE alkaline phosphatase conjugate (blue solution).

3.             Assay buffer: 1 vial containing 10.5 ml of ready-to-use yellow solution.

4.             Wash solution concentrate: 1 vial of 10 ml of concentrate (blue solution), to be diluted into 500 ml of distilled water and stored at 4°C.

5.                Microplate: 12 green color-coded strips. Break-apart wells are coated with Mouse monoclonal anti-h-IgE.

6.                Substrate diluent: 1 vial containing 10.5 ml of pNPP diluent.

7.                Chromogen tablets: 3 packs containing each 1 tablet of 5mg of pNPP (p-nitrophenyl phosphate).

8.             Stop solution: 1 vial containing 10.5 ml of NaOH 1N.

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 pipettors.

2.             Microplate reader with 405 nm filter.

3.             Incubator.

4.             Horizontal shaker.

5.             Microplate washer.

6.             Glass vials.

7.             Distilled water.

8.             Microwells extra holder frame.

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 wells for blank, standards, control sera and unknowns.

1.             Pipet 100 μl of assay buffer into each well.

2.             Pipet 20 μl of standards, control and samples into the corresponding wells.

 3.            Cover with plastic film, homogenize at 300 rpm (horizontal shaking) and incubate all strips in an incubator for 90 minutes at 37°C.

 4.            Aspirate and add 300 μl of wash solution to each well. Aspirate again. Repeat twice more for a total of 3 washes/aspirations. Allow the wells to stand for 1 minute, and aspirate again.

 5.            Pipet 100 μl of conjugate into each well.

 6.            Cover with plastic film and incubate all strips in an incubator for 90 minutes at 37°C.

 7.            30 minutes before step 8, prepare the chromogen-substrate solution. In an empty glass vial, dilute 1 tablet of pNPP into 3.3 ml of ready-to-use substrate diluent. Keep it in the dark. Use only fresh solution for each run.

 8.            Repeat wash procedure as described in step 4.

 9.            Pipet 100 μl of chromogen into each well.

10.           Cover with plastic film and incubate all strips for 30 minutes at room temperature in the dark.

11.           Pipet 100 μl of stopping solution (NaOH) into each well.

12.                Homogenize at 300 rpm (horizontal shaking) and read all wells at 405 nm against the blank, set at zero.

Total IgE microplates Flow Chart.

Data table (Example).

Calculation of results:

% B/Bmax = Mean O.D.(Stds, Control or samples) x 100

                                Mean O.D. Std. 500

Plot % B/Bmax 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.

Expected Values:

The values reported 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).