Epstein Barr Virus EA(D) IgG ELISA Kit

Cat. No.:DEIA3372
Pkg.Size:96T

Intended use
Epstein Barr Virus EA(D) IgG ELISA Kit is an ELISA kit for detection of IgG antibodies to diffusion component of an early antigen
(EA (D)) Epstein-Barr virus (EBV).

General Description
In 1961 an infectious disease was identified in Uganda, which was correlated with the appearance of a defined type of tumor
with children. The illness, which is found predominantly in Africa and Papua-New Guinea, was named Burkitt lymphoma from its
discoverer. In 1964, Epstein, Barr and Achong characterized by electron microscopy as the causing agent a hitherto unknown
virus, which belongs to the family of herpes viruses. The Epstein Barr virus is made responsible for a variety of diseases like
infectious mononucleosis, Burkitt lymphoma, as well as nasopharyngeal carcinoma. In addition, a role of the virus is discussed in
connection with Hodgkin´s disease. Especially with teenagers there appears a glandular fever syndrome, which is called "kissing
disease“. Diseases which are caused by the Epstein Barr virus are found mainly in persons with reduced immunity. For example,
the virus is associated with a lymphoproliferative disease which occurs after transplantation. The immune system of such
patients is usually impaired by drug therapy. Also in immune-deficient AIDS patients, there appears frequently a state where
cells at the rim of the tongue are infected (oral hairy leukoplakia). Infected persons keep the Epstein-Barr virus forever in their
body, they are however mostly not ill. In the developing countries practically all the people are infected, in the western world the
incidence is between 80% and 90%. The transmittance occurs already during childhood, perhaps by transfer from the mother,
mainly via the saliva. During the active phase of the viral cycle, the Epstein-Barr virus produces about 100 different antigens, in
the inactive phase around 10. The latter comprises among others the EBV nuclear antigen EBNA-1, which is closely correlated
with a past infection and an immunity. The early antigen (EA) as well as the virus capsid antigen (VCA) from the active phase
are also used as diagnostic markers.

Principle Of The Test

Epstein Barr Virus EA(D) IgG ELISA Kit is a solid-phase immunoanalytical test. The polystyrene strips are coated with a
recombinant antigen that bears immunodominant epitopes of EA protein. The anti-EA (D) antibodies, if present in the serum
(plasma), bind to the immobilized antigens. The antibodies that do not bind are washed away and those that formed complexes
with the antigens are later on recognised by animal anti-human IgG antibodies labelled with horseradish peroxidase. The
presence of labelled antibodies is revealed by an enzymatic reaction with a chromogenic substrate. Negative sera do not react
and the mild change in colour, if present, may be attributed to the reaction background.

Reagents And Materials Provided

1. ELISA break-away strips (green) coated with the recombinant antigen 1 microplate
2. Calibrator, r.t.u. 1) 1.3 mL
3. Negative control serum, r.t.u 1.3 mL
4. Positive control serum, r.t.u 1.3 mL
5. Anti-human IgG antibodies labelled with horseradish peroxidase, Px-conjugate 15 mL
6. Wash buffer concentrate, 10x concentrated 125 mL

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7. Dilution buffer (DIL), r.t.u. 1) 125 mL,
8. Chromogenic substrate (TMB substrate), r.t.u. 1) 15 mL
9. Stop solution, r.t.u. 1) 15 mL
10. Sealable pouch for unused strips
* (read to use)

Materials Required But Not Supplied

1. Distilled or deionized water for dilution of the washing solution.
2. Appropriate equipment for pipetting, solution dispensing and washing.
3. Spectrophotometer / colorimeter (microplate reader – wavelenght 450 nm).

Storage
Store the kit and the kit reagents at 2 to 10℃, in a dry place and protected from the light. Store unused strips in the sealable
pouch and keep the desiccant inside. Store serum samples at 2 to 10℃ up to one week. For longer period make aliquots and
keep them at -20℃. Avoid repeated thawing and freezing.

Specimen Collection And Handling

Vortex samples, Calibrator and the controls in order to ensure homogeneity and mix all solutions well prior use.
Dilute serum samples 1:100 in Dilution buffer and mix (e.g. 5 µL of serum sample + 500 µL of Dilution buffer). Do not dilute the
control sera and Calibrator, they are ready to use.

Reagent Preparation
1. Allow all kit components to reach room temperature.
2. Prepare Wash buffer by diluting the Wash buffer concentrate 10 times with an appropriate volume of distilled or deionised
water (e.g. 100 mL of the concentrated Wash buffer + 900 mL of distilled water). If there are crystals of salt present in the
concentrated Wash buffer, warm up the vial to 32 to 37℃ in a
3. Do not dilute Px-conjugate, TMB substrate and Stop solution, they are ready to use.

Assay Steps
1. Allow the microwell strips sealed inside the aluminium bag to reach room temperature. Withdraw an adequate number of
strips and put the unused strips into the provided pouch and seal it carefully with the desiccant kept inside.
2. Start with filling the first well with 100 µL of Dilution buffer (DIL) to estimate the reaction background. Fill next two wells with
100 µL/well of Calibrator (CAL) and then fill next two wells with Negative control serum (NCS). Fill the remaining wells with 100
µL of diluted serum samples (S1, S2, S3,...). It is also suitable to apply Positive control serum for the test control. It is sufficient
to apply serum samples as singlets, however, if you wish to minimize the laboratory error apply the samples and controls as
doublets, Calibrator as triplet.
3. Incubate 30 minutes (±2 min) at room temperature.
4. Aspirate the liquid from wells into a waste bottle containing an appropriate disinfectant. Wash and aspirate the wells four times
with 250 µL/well of Wash buffer. Avoid cross-contamination between wells!
If any liquid stays trapped inside the wells, invert the plate and tap it on an adsorbent paper to remove the remaining drops.
5. Shake the bottle with Anti-human IgG Px-conjugate and add 100 µL of diluted Px-conjugate into each well.
6. Incubate 60 minutes (±5 min) at room temperature.
7. Aspirate and wash four times with 250 µL/well of Wash buffer.
8. Dispense 100 µL of TMB substrate into each well.

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45-16 Ramsey Road Shirley, NY 11967, USA
Tel: 631-624-4882 ·Fax:631-614-7828
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9. Incubate for 10 minutes (+/-5 seconds) at room temperature.
The time measurement must be started at the beginning of TMB dispensing. Cover the strips with an aluminium foil or keep
them in the dark during the incubation with TMB substrate.
10. Stop the reaction by adding 100 µL of Stop solution. Use the same pipetting rhythm as with the TMB substrate to ensure the
same reaction time in all wells. Tap gently the microplate few times to ensure complete mixing of the reagents.
11. Measure the absorbance at 450 nm with a microplate reader within 20 minutes. It is recommended to use reference reading
at 630 nm.

Evaluation
Qualitative evaluation
1. Compute the absorbance mean of Calibrator.
2. Compute the cut-off value by multiplying the Calibrator mean with a Correction factor. The Correction factor value for
particular Lot is written in Quality control certificate.
3. Samples with absorbances lower than 90% of the cut-off value are considered negative and samples with absorbances higher
than 110% the cut-off value are considered positive.
Semiquantitative evaluation
Determine the Positivity Index for each serum sample as follows:
1. Compute the cut-off value using the Calibartor mean and the Corection factor (see the previous paragraph).
2. Compute the Positivity Index for each sample according to the following formula:
Sample positivity Index = (Sample absorbance)/(Cut – off value)

Recovery
Measured values of recovery test for every Lot are between 80-120% of expected values.

Reproducibility
intra-assay: 3.7-3.8%.
Inter-assay: 4.7-8.7%.

Interferences
Haemolytic and lipemic samples have no influence on the test results up to concentration of 50 mg/mL of haemoglobin, 5 mg/mL
of bilirubin and 100 mg/mL of triglycerides.

Precautions
1. Calibrator and Controls contain human sera that has been tested negative for HBsAg, anti-HIV-1,2 and anti-HCV. However
they should be regarded as contagious and handled and disposed of according to the appropriate regulations.
2. Autoclave all reusable materials that were in contact with human samples for 1 hour at 121℃, burn disposable ignitable
materials, decontaminate liquid wastes and nonignitable materials with 3% chloramine. Liquid wastes containing acid (Stop
solution) should be neutralized in 4% sodium bicarbonate solution.
3. Handle Stop solution with care. Avoid contact with skin or mucous membranes. In case of contact with skin, rinse immediately
with plenty of water and seek medical advice.
4. Do not smoke, eat or drink during work. Do not pipette by mouth. Wear disposable gloves while handling reagents or samples
and wash your hands thoroughly afterwards. Avoid spilling or producing aerosol.

REFERENCES

Creative Diagnostics. All rights reserved

45-16 Ramsey Road Shirley, NY 11967, USA
Tel: 631-624-4882 ·Fax:631-614-7828
E-mail: info@creative-diagnostics.com
www.creative-diagnostics.com

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1. Dolken G., Weitzmann U., Boldt C., Bitzer M., Brugger, Lohr W.: Enzyme-linked Assay for IgG Antibodies to Epstein Barr Virus
– Associated Early Antigens and Viral Capsid Antigen. J.Immunol.Meth.67:225-233 (1984)
2. Karray H., Ayadi W., Fki L., Hammami A., Daoud J., Drira MM, Frikha M., Jlidi R., Middeldorp JM: Comparison of three
different serological techniques for primary diagnosis and monitoring of nasopharyngeal carcinoma in two age groups from
Tunisia: J.Med.Virol.75(4): 593-602, (2005)
3. Henle W. & Henle G.: Seroepidemiology of the virus. In: The Epstein-Barr virus, pp. 61-73, eds. Epstein M. A. & Achong B.G.,
Springer, Berlin, 1979.

Creative Diagnostics. All rights reserved

45-16 Ramsey Road Shirley, NY 11967, USA
Tel: 631-624-4882 ·Fax:631-614-7828
E-mail: info@creative-diagnostics.com
www.creative-diagnostics.com