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DIAGNOSTIC AUTOMATION, INC.

23961 Craftsman Road, Suite D/E/F, Calabasas, CA 91302


Tel: (818) 591-3030 Fax: (818) 591-8383
onestep@rapidtest.com
technicalsupport@rapidtest.com
www.rapidtest.com

See external label 2°C-8°C Σ=96 tests Cat # 1415Z

EBV-EA IgG
Cat # 1415Z

Cat # Number 1415Z

Test EBV -EA IgG ELISA


Method ELISA: Enzyme Linked Immunosorbent Assay
Principle ELISA - Indirect; Antigen Coated Plate

Detection Range Qualitative Positive; Negative control & Cut off

Sample 5ul Serum


Specificity 100 %
Sensitivity 100 %

Total Time ~90 min

Shelf Life 12 -18 Months

* Laboratory results can never be the only base of a medical report. The patient history and further tests have to be
taken into account

DAI Code # 11 1
NAME AND INTENDED USE
The DIAGNOSTIC AUTOMATION Epstein Barr Virus Early Antigen (EBV-EA) IgG Enzyme-linked
Immunosorbent Assay (ELISA), is intended for the detection of IgG antibody to Epstein Barr Virus Nuclear
Antigen-1 in human sera and plasma.

SUMMARY AND EXPLANATION OF THE TEST


Detection of the Epstein-Barr virus was first described in 1964 by Epstein, Achong, and Barr using electron
microscopic studies of cultured lymphoblasts derived from patients with Burkitt’s lymphoma1. EBV is classified
as a member of the herpes-virus family based upon it’s characteristic morphology2,3.
EBV infection may demonstrate a wide spectrum of clinical symptoms. The majority of primary EBV infections
are transmitted via saliva, occur during childhood, and are subclinical4. Antibody titers to specific EBV antigens
correlate with different stages of IM. Both IgM and IgG antibodies to the viral capsid antigen (VCA) peak 3 to 4
weeks after primary EBV infection. IgM anti-VCA declines rapidly and is usually undetectable after 12 weeks.
IgG anti-VCA titers decline slowly after peaking but last indefinitely. Antibodies to EBV nuclear antigen
(EBNA) detected by anticomplement immunofluorescence develop from 1 month to 6 months after infection; and,
like anti-VCA, persist indefinitely6. Antibodies to EBNA indicate that the EBV infection was not recent. EBV
early antigen (EA) consists of two components; diffuse (D), and restricted (R). The terms D and R reflect the
different patterns of immunofluorescence staining exhibited by the two components. Antibodies to EA may
appear transiently for up to three months or longer during the acute phase of IM in 85% of patients7. The
antibody response to EA in IM patients is usually to the D component, whereas silent seroconversion to EBV in
children may produce antibodies to the R components. A definitive diagnosis of primary EBV infection can be
made with 95% of acute phase sera based on antibody titers to VCA, EBNA, and EA7.
Antibodies to EA, usually to the R component, together with antibodies to EBNA and high titers of IgG anti-
VCA, may be associated with reactivation of the latent viral carrier state. EBV positive serology associated with
reactivation of EBV is found in sera of patients with immunodeficiencies8, patients with recurrent parotitis9,

immunosuppressed patients, pregnant women, and persons of advanced age. Antibodies to the R component may
be found at moderate to high levels in patients with Burkitt’s lymphoma. In contrast, patients with
nasopharyngeal carcinoma may produce high titer antibodies to the D component. Elevated levels of anti-EA and
IgG anti-VCA may be detected in patients with chronic or recurrent illness suspected of being caused by EBV8.
However, a diagnosis of chronic EBV should not be based on the presence of antibodies to EA since elevated
anti-EA titers may also be found in patients with other diseases as well as in healthy individuals with past EBV
infections 5.

PRINCIPLE OF THE TEST


Purified EBV-EA antigen is coated on the surface of microwells. Diluted patient serum is added to wells, the
anti- EBV-EA specific antibody, if present, will bind to the antigen. All unbound materials are washed away.
After adding enzyme conjugate, it binds to the antibody-antigen complex. Excess enzyme conjugate is washed
off, and TMB Chromogenic substrate is added. The enzyme conjugate catalytic reaction is stopped at a specific
time. The intensity of the color generated is proportional to the amount of specific antibody in the sample. The
results are read by a microwell reader compared in a parallel manner with calibrator and controls.

DAI Code # 11 2
MATERIAL PROVIDED
1. Microwell strips: EBV-EA antigen coated wells. (12 x 8 wells)
2. Sample Diluent: Blue Color solution 1 vial (22 ml)
3. Calibrator: Factor value (f) stated on label. Red Cap. 1 vial (150 µl)
4. Negative control: Range stated on label. Natural Cap. 1 vial (150 µl)
5. Positive control: Range stated on label. Green Cap. 1 vial (150 µl)
6. Washing concentrate 10x: White Cap. 1bottle (100 ml)\
7. Enzyme conjugate: Red color solution. 1 vial (12 ml)
8. TMB Chromogenic Substrate: Amber bottle. 1 vial (12 ml)
9. Stop solution: 2 N HCl. 1 vial (12 ml)

STORAGE AND STABILITY


1. Store the kit at 2 - 8o C.
2. Always keep microwells tightly sealed in pouch with desiccants. We recommend you use up all wells within
4 weeks after initial opening of the pouch.
3. The reagents are stable until expiration of the kit.
4. Do not expose test reagents to heat, sun, or strong light during storage or usage

WARNINGS AND PRECAUTIONS


1. Potential biohazardous materials:
The calibrator and controls contain human source components which have been tested and found nonreactive
for Hepatitis B surface antigen as well as HIV antibody with FDA licensed reagents. However, as there is no
test method that can offer complete assurance that HIV, Hepatitis B virus, or other infectious agents are
absent, these reagents should be handled at the Biosafety Level 2, as recommended in the Centers for Disease
Control / National Institutes of Health manual, “Biosafety in Microbiological and Biomedical Laboratories.”
1984
2. Do not pipette by mouth. Do not smoke, eat, or drink in the areas in which specimens or kit reagents are
handled.
3. The components in this kit are intended for use as an integral unit. The components of different lots should
not be mixed.
4. This product contains components preserved with sodium azide. Sodium azide may react with lead and
copper plumbing to form explosive metal azide. On disposal, flush with a large volume of water.

SPECIMEN COLLECTION AND HANDLING


1. Collect blood specimens and separate the serum.
2. Specimens may be refrigerated at 2 - 8o C for up to seven days or frozen for up to six months. Avoid
repetitive freezing and thawing of serum sample.

PREPARATION FOR ASSAY


1. Prepare 1x washing buffer.
Prepare washing buffer by adding distilled or deionized water to 10x wash concentrate to make a final volume
of 1 liter.
2. Bring all specimens and kit reagents to room temperature (20 - 25o C) and gently mix

DAI Code # 11 3
ASSAY PROCEDURE
1. Place the desired number of coated strips into the holder.
2. Prepare 1:20 dilutions by adding 10 µl of the samples, negative control, positive control, and calibrator to
200 µl of sample diluent. Mix well.
3. Dispense 100 µl of diluted sera, calibrator, and controls into the appropriate wells. For the reagent blank,
dispense 100 µl sample diluent in 1A well position. Tap the holder to remove air bubbles from the liquid and
mix well. Incubate for 30 minutes at room temperature.
4. Remove liquid from all wells. Repeat washing three times with washing buffer.
5. Dispense 100 µl of enzyme conjugate to each well and incubate for 30 minutes at room temperature.
6. Remove enzyme conjugate from all wells. Repeat washing three times with washing buffer.
7. Dispense 100 µl of TMB Chromogenic Substrate to each well and incubate for 30 minutes at room
temperature.
8. Add 100 µl of 2 N HCl to stop reaction.
Make sure there are no air bubbles in each well before reading.
9. Read O.D. at 450 nm with a microwell reader.

CALCULATION OF RESULTS
1. To obtain Cut off OD value: Multiply the OD of Calibrator by Factor (f) printed on
label of Calibrator.
2. Calculate the EBV-EA IgG Index of each determination by dividing the OD values
of each sample by obtained OD value of Cut off.

For example:
If Factor (f) value on label = 0.4
Obtained Calibrator O.D. = 1.100
Cut-off O.D. = 1.100 x 0.4 = 0.44 (By definition EBV-EA IgG Index = 1)
Patient sample O.D. = 0.580
EBV-EA IgG Index = 0.580 / 0.44 = 1.32 (Positive result)
Patient sample O.D. = 0.320
EBV-EA IgG Index = 0.320 / 0.44 = 0.73 (Negative result)

QUALITY CONTROL
The test run may be considered valid provided the following criteria are met:
1. The O.D. value of the reagent blank against air from a microwell reader should be less than 0.150.
2. If the O.D. value of the Calibrator is lower than 0.250, the test is not valid and must be repeated.
3. The EBV-EA IgG Index for Negative and Positive Control should be in the range stated on the labels.

INTERPRETATION

Negative: EBV-EA IgG Index of 0.90 or less.


Equivocal: EBV-EA IgG Index of 0.91 - 0.99 are equivocal. Sample should be retested.
Positive: EBV-EA IgG Index of 1.00 or greater.

DAI Code # 11 4
PERFORMANCE CHARACTERISTICS
Histogram:
215 random samples are determined with DIAGNOSTIC AUTOMATION Microwell ELISA EBV-EA IgG. The
test results are computed as IgG Index using a chosen reference serum as IgG Index 1. The distribution of
frequency versus IgG Index value is presented as following:

P / N Ratio
63.2% (136 samples) have IgG index below 1.
Mean value = 0.509 SD = 0.229
IgG index 1 (cut off value) = Mean value + 2 SD

36.7% (79 samples) have IgG index greater than 1.


Mean value = 2.352 SD = 1.324
P / N ratio = Mean of POSITIVE / Mean of NEGATIVE
= 2.352 / 0.509 = 4.6

Expected Values and Prevalence:


215 specimens from random asymptomatic blood donors were tested with the DIAGNOSTIC AUTOMATION
EBV-EA IgG ELISA. 79 were found to be positive (36.7 %) and 136 were found to be negative (63.2 %).
Prevalence may vary depending on a variety of factors such as geographical location, age, socioeconomic status,
race, type of test employed, specimen collection and handling procedures, clinical and epidemiological history.

Precision:
The precision of the assay was evaluated by testing three different sera of eight replicates over a period of one
week. The intra-assay and inter-assay C.V. are summarized below:

Negative Low positive Positive


Intra-assay 12.5% 8.5% 5.6%
Inter-assay 14.8% 10.9 % 8.5%

DAI Code # 11 5
LIMITATIONS OF THE ASSAY
1. The values obtained from this assay are intended to be an aid to diagnosis only. Each physician must interpret
the results in light of the patient’s history, physical findings and other diagnostic procedures.
2. Results from children should be reviewed with caution. This kit is designed to measure IgG antibody in
patient samples. Positive results in neonates must be interpreted with caution, since maternal IgG is
transferred passively from the mother to the fetus before birth.
3. Results obtained from immunocompromised individuals should be interpreted with caution.
4. There is a possibility of assay cross-reactivity with specimens containing anti-E.coli antibody.

REFERENCES
1. Epstein, M.A., B.B. Achong, and Y.M. Barr. 1964. Virus particles in Cultured Lymphoblasts from Burkitt’s
Lymphoma. In: Lancet 1:702-703.
2. Epstein, M.A., Y.M. Barr, and B.G. Achong. 1965. Studies with Burkitt’s Lymphoma. In: Wistar Inst.
Sympos. Monogr. 4:69-82.
3. Schooley, R.T. and R. Dolin. 1985. Epstein-Barr Virus Infectious Mononucleosis). In: Principles and
Practice of Infectious Diseases, 2nd Edition. Mandell, G.L., R.G. Douglas, and J.E. Bennett. (eds). John
Wiley and Sons, New York. pp 97-982.
4. Lennette, E.T. 1988. Herpesviridae: Epstein-Barr Virus. In: Laboratory Diagnosis of Infectious Diseases,
Principles and Practice. Vol. Ll. Lennette, E.H., Halonen, P., Murphy, F.A., eds. Springer-Verlag, NY. Pp.
230-246.
5. Lennette, E.T. and W. Henle. 1987. Epstein-Barr Virus Infections: Clinical and Serological Features.
Laboratory Management June: pp. 22-28.
6. Henle G., W. Henle, and C.A. Horwitz. 1974. Antibodies to Epstein-Barr Virus-Associated Nuclear Antigen
in Infection Mononucleosis. Journal of infectious Diseases. 130:231-239.
7. Lennette ET, and Henle W: Epstein-Barr virus infections: Clinical and Serologic features. Lab Mgmt. 23-28,
June, 1987.
8. Joncas J, Lapointe N, Gervais F, Leyritz M, and Wills A: Unusual prevalence of antibodies to Epstein-Barr
virus early antigen in ataxia telangiectasia. Lancet 1:1160, 1977.
9. Akaboshi I, Jamamoto J, Katsuki T, and Matsuda l: Unique pattern of Epstein-Barr virus specific antibodies
in recurrent parotitis. Lancet 2:1049-1051, 1983.

Date Adopted Reference No.


2011-04-14 DA-EBV-EA IgG-2011

DIAGNOSTIC AUTOMATION, INC.


23961 Craftsman Road, Suite D/E/F, Calabasas, CA 91302
Tel: (818) 591-3030 Fax: (818) 591-8383
ISO 13485-2003

Revision Date: 04-19-2011

DAI Code # 11 6

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