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Enzyme immunoassays (microtiter strips) for the quantitative determination of
IgA, IgG or IgM antibodies against the „early antigen“ (EA) of Epstein Barr Virus

The Epstein Barr Virus (EA) IgA/IgG/IgM Enzyme Immunoassay Kits provide materials for the quantitative determination of human IgA, IgG or IgM antibodies against the EBV early antigen (EA) in serum and plasma. This assay is intended for in-vitro diagnostic use only.

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 ist 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.

In a fresh infection, IgM antibodies against VCA and EA are determined by immunofluo-rescence or ELISA. Later VCA IgG and afterwards EBNA-1 IgG antibodies appear. The simultaneous activation of VCA IgM and EBNA-1 IgG indicates correspondingly a reactivation of a latent EBV infection.

The IBL EBV (EA) IgM ELISA is an excellent tool to detect fresh infections. With the IBL EBV (EA) IgG ELISA an optimal monitoring of convalescence and reactivated infections as well as the detection of nasopharynx carcinoma and Burkitt Lymphoma is possible. Reliable diagnosis of immune response at nasopharynx carcinoma and chronic reactivated EBV infections guarantees the IBL EBV (EA) IgA ELISA.

The Epstein Barr Virus (EA) IgA/IgG/IgM ELISAs are based on the principle of the enzyme immunoassay (EIA). Affinity-purified, natural antigen from the human Burkitt Lymphoma cell line P3H3 is bound on the surface of the microtiter strips. Diluted patient serum or ready to use standards and controls are pipetted into the wells of the microtiter plate. A binding between the IgA/IgG/IgM antibodies of the serum and the immobilized antigen takes place. After a one hour incubation at room temperature, the plate is rinsed with diluted wash solution, in order to remove unbound material. Then ready to use anti human IgA, IgG or IgM peroxidase conjugate is added and incubated for 30 minutes. After a further washing step, the substrate (TMB) solution is pipetted and incubated for 20 minutes, inducing the development of a blue dye in the wells. The colour development is terminated by the addition of a stop solution, which changes the colour from blue to yellow. The resulting dye is measured spectrophotometrically at the wavelength of 450 nm. The concentration of the IgA/IgG/IgM antibodies is directly proportional to the intensity of the colour.

· The assay calibrators and controls are of human origin and have been tested and confirmed negative for HIV I/II, HBsAg and HCV by FDA approved procedures. All standards, however, should be treated as potential biohazards in use and for disposal.

· The assay reagents contain sodium azide or thimerosal which may be toxic if ingested. Sodium azide may react with copper and lead piping to form highly explosive salts. On disposal, flush with large quantities of water.

· The stop solution contains H₂SO₄. If it comes into contact with skin, wash thoroughly with water and seek medical attention. Since the H₂SO₄ used to terminate the colour reaction is corrosive, the instrumentation employed to dispense it should be thoroughly cleaned after use.

· Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes. If contact occurs, wash with a germicidical soap and copious amounts of water.

· Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.

· Wear disposable latex gloves when handling specimens and reagents, and wash hands thoroughly afterwards. Microbial contamination of reagents or specimens may give false results.

· Handling should be in accordance with the procedures defined by an appropriate national biohazard safety guideline or regulation.

· Chemicals and prepared or used reagents have to be treated as hazardous waste according the national biohazard safety guideline or regulation.

· In case of any damage of the kit or kit components, IBL have to be informed written, latest 1 week after receiving the kit.

· On request safety data sheets are at your´s disposal. The safety data sheets fits the demands of:

Store all reagents at 2 - 8 °C and use before expiry date. When stored at 2 - 8 °C unopened reagents will retain reactivity until expiration date. Do not use reagents beyond this date.

Once the foilbag of the coated microtiter strips has been broken, care should be taken to close it tightly again. The immuno reactivity of the coated microtiter strips are stable for approx. 3 months in the broken, but tightly closed bag when stored at 2 - 8 °C.

Allow all reagents and required number of strips to reach room temperature prior to use.

Standard		A	B	C	D
	IgA	1	10	35	200
Concentration in U/ml	IgG	1	10	50	150
	IgM	1	10	35	200

Standard B = Cut-Off Standard

· Automatic pipettes to dispense 5, 50, 100 and 500 µl (a multichannel pipetting device such as Titertek is suitable for adding reagents to the wells)

Serum or plasma (EDTA, heparin) should be used, and the usual precautions for venipuncture should be observed. No special sample pretreatment is necessary. The specimen may be stored at 2 - 8 °C for up to 48 hours, and should be frozen at - 20 °C or lower for longer periods.

Samples suspected to contain concentrations higher than the highest standard have to be diluted further with sample diluent buffer.

Dilute patient sample 1 to 101 with ready to use sample diluent (e. g. 5 µl sample + 500 µl buffer).

In order to avoid interference of rheumatoid factors, patient sera should be treated with RF absorbant (IBL Cat. No. RE 590 59). Alternatively, positive results can be confirmed in a second test run. Do not treat the controls!

Dilute the wash buffer concentrate with distilled water 1 to 10 (1 + 9)(e. g. 10 ml concentrate + 90 ml distilled water.). If during the cold storage crystals precipitate, the concentrate should be warmed up at 37 °C for15 minutes. ). Ready to use wash buffer is stable for at least 8 weeks when stored at 2 - 8 °C.

All reagents and specimens must be allowed to come to room temperature before use. All reagents must be mixed without foaming.Once the test has been started, all steps should be completed without interruption.Use new disposable plastic pipette tips for each reagent, standard or specimen in order to avoid cross contamination. For the dispensing of the TMB substrate solution and the TMB stop solution avoid pipettes with metal parts.Absorbance is a function of the incubation time and temperature. Before starting the assay, it is recommended that all reagents be ready, caps removed, all needed wells secured in holder, etc. This will ensure equal elapsed time for each pipetting step without interruption.

8.1. Leave sufficient microtiter strips in the strip holder to enable the running of standards and samples. Secure the desired number of microtiter strips in the holder.

8.2. Pipet 100 µl of standards and diluted samples into the appropriate wells of the strips.

8.3. Cover plate with the enclosed foil and incubate for 60 minutes at room temperature (18 - 24 °C).

8.4. Washing: discard the incubation solution, rinse the wells 3 x with 300 µl wash buffer (dilute concentrate 1 to 10 with distilled water) and remove any residual.

8.6. Cover plate with the enclosed foil and incubate for 30 minutes at room temperature (18 - 24 °C).

8.7. Washing: discard the incubation solution, rinse the wells 3 x with 300 µl wash buffer (dilute concentrate 1 to 10 with distilled water) and remove any residual.

8.8. Promptly pipet 100 µl of the TMB substrate solution into the rinsed wells.

8.9. Cover plate with the enclosed foil and incubate for 20 minutes at room temperature (18 - 24 °C) in the dark.

8.10. Stop the reaction by adding 100 µl of TMB stop solution to each well.

8.11. Shake gently the Microtiter Strips being careful not to let the content come from the wells and read at 450 nm within 60 minutes from the stopping.

The ready to use standards of the Epstein Barr Virus (EA) IgA/IgG/IgM ELISA are defined and expressed in Units (U). This results in an exact and reproducible quantitative evaluation. Consequently for a given patient follow-up controls become possible.

For a quantitative evaluation the absorptions of the standards are graphically drawn against their concentrations. From the resulting reference curve the concentration values for each patient sample can then be extracted in relation to their absorptions. The initial dilution of 1:101 has already been reconsidered.

Alternatively the use of electronic device is possible. The results can also be calculated with normal programs for automatic data processing, i.e. 4 parameter, spline, logit-log.Any sample reading greater than the highest standard should be diluted appropriately with sample diluent buffer and reassayed. The result has to be multiplied by the additional dilution factor.

Do not use the following calibration curve. In the laboratory the standard curve should be established in each assay run.

Standards (U/ml)	OD 450 nm
1	0.009
10	0.475
50	1.152
150	1.842

> 12 U/ml	positive
8 - 12 U/ml	borderline
< 8 U/ml	negative

The calculated absorptions for the patient sera, as mentioned above, are compared with the value for the cut-off standard. If the value of the sample is higher, there is a positive result. For a value below the cut-off standard, there is a negative result. It seems reasonable to define a range of +/- 20 % around the value of the cut-off as a grey zone. In such a case the repetition of the test with the same serum or with a new sample of the same patient, taken after 2-4 weeks, should be recommended. Both samples should be measured in parallel in the same run.

The positive control must show at least the double absorption compared with the cut-off standard.

The intra-assay coefficient of variation of the Epstein Barr Virus (EA) IgA/IgG/IgM ELISA was assessed by a ten-fold determination in a positive serum sample to less than 10 %.

Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the package insert instructions and with adherence to good laboratory practice.

Azide and thimerosal at concentrations higher than 0.1 % interfere in this assay. Therefore control sera or samples containing higher concentrations of the above mentioned components may give false results.

Reagents from different kits or lots should not be mixed, due to possible different shipping or storage conditions.

Any improper handling of samples or modification of this test might influence the results. Interferences caused by improper sample handling are explained in chapter ‘Specimen Collection and Storage’.

For diagnostic purpose results obtained by this assay should be used in conjunction with other test results, the overall clinic presentation to the physician, and all other appropriate information.

It is recommended to use control samples according to state and federal regulations. The use of control samples is advised to assure the day to day validity of results. Use controls at both normal and pathological levels.

The controls and the corresponding results of the IBL QC-Laboratory are stated at the QC certificate added to the kit.

Employ appropriate statistical methods for analysing control values and trends. If the results of the assay does not comply with the established limits and repetition excludes errors in technique, patient sample should be considered invalid. Check the following areas:

Pipetting and timing devices; photometer, expiration dates of reagents, storage and incubation conditions, aspiration and washing methods

After checking the above mentioned items without finding any error contact your distributor or IBL directly.

Any modification of this test as well as exchange or mixture of any components from different lots might influence the results. In such cases there is no claim for a replacement.

· Wash Buffer: Dilute 1:10 with distilled water (e.g. 10 ml concentrate + 90 ml dist. water). Store at 2 – 8 °C for max. 8 weeks.

2. Specimen Collection and Storage Samples: Serum, plasma. Storage for up to 2 days at 2 - 8 °C, longer storage at –20°C. Avoid repeated freezing and thawing.

Dilute samples 1:101 with sample diluent (e.g. 5 µl sample + 500 µl sample diluent).

For IgM: In order to avoid interference of rheumatoid factors, patient sera should be treated with RF absorbant (IBL Cat. No. RE 590 59). Do not treat the standards/controls!

3. Assay Procedure Storage of reagents at 2 - 8 °C. Allow reagents to reach room temperature.

** Stop solution should be pipetted in the same time schedule as substrate solution.

Patient results are read directly from the graph constructed from the standards, the initial sample dilution (1:101) has already been reconsidered. Samples with values above the highest standard have to be diluted further. In this case, the additional dilution factor has to be taken into account.

Epstein Barr Virus (EA)

Cat.-No. : IgA: RE 562 11

IgG: RE 562 21

IgM: RE 562 31

Size:12 x 8(Break Apart) Storage:2 - 8 °C

Standard B = Cut-Off Standard