BIOTEST ANTI-EBV RECOMBINANT, EBNA IGG

K983841 · Biotest Diagnostics Corp. · LSE · Jun 9, 1999 · Microbiology

Device Facts

• Record ID: K983841
• Device Name: BIOTEST ANTI-EBV RECOMBINANT, EBNA IGG
• Applicant: Biotest Diagnostics Corp.
• Product Code: LSE · Microbiology
• Decision Date: Jun 9, 1999
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3235
• Device Class: Class 1
• Attributes: Pediatric

Intended Use

The Biotest EBNA IgG ELISA is an enzyme immunoassay using a recombinant antigen for the qualitative detection of IgG antibodies to the Epstein-Barr Virus (EBV) EBNA-1 (Nuclear Antigen 1) in human serum or plasma. Results obtained with this test, in conjunction with other clinical and patient data obtained in assays for other Epstein-Barr antigens such as Early Antigen IgG and IgM, assist in serological diagnosis of EBV infection in pediatric and adult populations.

Device Story

Biotest Anti-EBV Recombinant EBNA IgG is an indirect ELISA test system for qualitative detection of IgG antibodies to EBV EBNA-1 in human serum or plasma. Device uses recombinant EBNA-1 p72 antigen immobilized on 96-well microtest plates. Patient serum/plasma is incubated; specific antibodies bind to antigen; non-specific antibodies are washed away. Murine monoclonal anti-human IgG antibody-enzyme-conjugate is added; final reaction converts colorless substrate to colored product. Color intensity measured via spectrophotometer at 450 nm; concentration relates to antibody levels. Used in clinical laboratories by trained personnel. Results assist clinicians in diagnosing EBV infection stages (acute, past, reactivation) when combined with other clinical data and EBV antigen assays. Benefits include improved diagnostic accuracy for EBV infection status.

Clinical Evidence

Clinical study of 408 patient samples (pediatric and adult, ages 1-74) across two sites. Evaluated against EBNA anti-complement immunofluorescence (ACIF) tests and clinical interpretation. Combined results vs ACIF: 86.8% sensitivity, 97.1% specificity. Clinical interpretation comparison showed 98.5% sensitivity and 98.6% specificity across various infection stages (acute, convalescent, past, reactivation, negative). Reproducibility evaluated via inter-run, intra-run, and inter-lab testing with C.V.s generally ranging from 4.8% to 19.4%. No cross-reactivity observed with HSV I/II, VZV, or CMV.

Technological Characteristics

ELISA-based immunoassay. Recombinant EBNA-1 p72 antigen immobilized on 96-well microtest plates. Detection via murine monoclonal anti-human IgG antibody-enzyme-conjugate (HRP). Chromogen: TMB. Readout: spectrophotometer at 450 nm. Specimen: serum or plasma. Storage: 2-8°C or -20°C.

Indications for Use

Indicated for use in pediatric and adult populations as an aid in the serological diagnosis of EBV infection, in conjunction with other clinical data and assays for other EBV antigens (Early Antigen IgG and IgM).

Regulatory Classification

Identification

Epstein-Barr virus serological reagents are devices that consist of antigens and antisera used in serological tests to identify antibodies to Epstein-Barr virus in serum. The identification aids in the diagnosis of Epstein-Barr virus infections and provides epidemiological information on diseases caused by these viruses. Epstein-Barr viruses are thought to cause infectious mononucleosis and have been associated with Burkitt's lymphoma (a tumor of the jaw in African children and young adults) and postnasal carcinoma (cancer).

Predicate Devices

• EBNA IgG ELISA Kit (K946158)

Submission Summary (Full Text)

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JUN 9 1999

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K983841

# ATTACHMENT I

REVISED 510(k) SUMMARY

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# 510(k) Summary of Information Respecting Safety and Effectiveness

- A. Name and Address of Submitter
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B.

| Company Name and Address: | Biotest Diagnostics Corporation<br>66 Ford Road, Suite 131<br>Denville, NJ 07834 |
|-----------------------------------|----------------------------------------------------------------------------------|
| Telephone: | [phone] |
| FAX: | [phone] |
| Contact Person: | Patricia E. Bonness, Official Correspondent |
| Date 510(k) Summary was Prepared: | October 26, 1998 |
| Device Names: | |
| Proprietary Name: | Biotest Anti-EBV Recombinant<br>EBNA IgG |
| Common Name: | EBNA IgG |
| Classification Name: | Epstein-Barr virus serological reagents |

# C. Legally Marketed Device

Biotest Diagnostics claims substantial equivalence to the EBNA IgG ELISA Kit (K946158) currently in commercial distribution by INCSTAR Corporation, Stillwater, MN.

# D. Device Description

Using recombinant DNA technology, Biotest has developed three highly purified EBV antigens for use in their ELISA test system.

- EBNA-1 p72: Major antigen of the EBNA complex. The recombinant protein does not contain the glycine-alanine copolymer, a structural feature of EBNA-1, which shows cross-reactivities with certain autoantibodies and CMV (IgM response).
- EA-D p64: Early antigen. Dominant immunogen of the EA-D complex.
- EA p138: Early antigen and major DNA binding protein. This highly reactive antigen is not detectable in EA immunofluorescence assays based on chemically induced Raji cells (deletion within the Raji genome).

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| Biotest<br>Anti-EBV<br>recombinant | recombinant antigens | | | monoclonal secondary antibody (HRP-conjugated) | |
|------------------------------------|----------------------|-----|------|------------------------------------------------|----------------|
| | p72 | p54 | p134 | anti-human IgG | anti-human IgM |
| EA IgM | | X | X | | X |
| EA IgG | | X | X | X | |
| EBNA IgG | X | | | X | |

#### Biotest Anti-EBV ELISA TESTS

The EBV immune status will be determined by the detection of specific antibodies directed against EBV proteins according to the principle of the indirect ELISA. The antigens are purified to apparent homogeneity and immobilized on the solid phase (microtest plate, 96 wells). If the patient's serum contains specific antibodies they will bind during the first incubation. Non-specific antibodies are removed by washing steps. During a second incubation the captured IgG antibodies are labeled. This is performed by addition of murine monoclonal anti-human IgG antibody-enzyme-conjugates. The final reaction converts a colorless substrate to a colored product. The concentration of color after a definite time is related to the concentration of antibody in the serum sample.

# E. Intended Use

The Biotest EBNA IgG ELISA is an enzyme immunoassay using a recombinant antigen for the qualitative detection of IgG antibodies to the Epstein-Barr Virus (EBV) EBNA-1 (Nuclear Antigen 1) in human serum or plasma. Results obtained with this test, in conjunction with other clinical and patient data obtained in assays for other Epstein-Barr antigens such as Early Antigen IgG and IgM, assist in serological diagnosis of EBV infection in pediatric and adult populations.

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- F. Comparison with Predicate Device
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A summary comparison of the features of the Biotest EBNA IgG and the INCSTAR EBNA IgG test kits is provided in Table 1 below:

# Table 1 Feature Comparison of Biotest and INCSTAR EBNA IgG Test Kits

| | Biotest | INCSTAR |
|------------------------------------------|--------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------|
| Intended Use | Detection of IgG antibodies<br>to EBV EBNA-1<br>Qualitative only | Detection of IgG antibodies<br>to EBV EBNA-1<br>Qualitative and semi-quantitative |
| Assay Method | ELISA | ELISA |
| Reactive Ingredients | Recombinant EBV (EBNA-1 p72)<br>Peroxidase-conjugated monoclonal<br>anti-human IgG (mouse) | EBNA-1 peptide<br>Peroxidase - conjugated<br>goat anti-human IgG |
| Specimen: Type<br>Min. Volume<br>Storage | Serum or plasma<br>25 μl<br>2 - 8°C or -20°C | Serum<br>50 μl<br>2 - 8°C/7days or -20°C |
| Controls | Negative<br>Positive | Negative<br>Low Positive<br>High Positive |
| Chromogen | TMB | TMB |
| Results:<br>Evaluation | 450 nm | 450 nm |
| (spectrophotometer) | | |
| Kit Size | 96 tests | 96 tests |

ਦੇ ਰੇ

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#### G. Performance Data

## Sensitivity/Specificity

The performance of the Biotest EBNA IgG ELISA was evaluated in a clinical study of 408 patient samples conducted at two geographically distinct locations. Samples were obtained from both pediatric and adult patients (ages 1 to 74) representing acute (139), late acute (34), recent past (12), past (120), reactivation (22), past/probable reactivation (11), and negative (70) disease stages of EBV infection.

Two methods were used to evaluate the performance of the Biotest EBNA IgG ELISA: direct comparison with commercially available or published EBNA anti-complement immunofluorescence (ACIF) tests, and comparison with clinical interpretation (stage of infection based on antibody patterns and clinical diagnosis at the time the specimen was drawn).

Results of the direct comparison with ACIF for both sites combined demonstrated a relative sensitivity of 86.8% and a relative specificity of 97.1%.

#### Table 1

# Clinical Site 1 Direct Comparison to ACIF

| EBNA ACIF | | | |
|-----------|-----|----|-------|
| | + | - | Total |
| Biotest + | 95 | 0 | 95 |
| Biotest - | 24 | 88 | 112 |
| Total | 119 | 88 | 207 |

Relative Sensitivity = 79.8% Relative Specificity = 100% Relative Agreement = 88.4% (C.I. = 72.6 to 87%) (C.I. = 95.9 to 100%)

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| | T | 'able | 2 |
|--|---|-------|---|
|--|---|-------|---|

| | Clinical Site 2 | | |
|---------------------------|-----------------|--|--|
| Direct Comparison to ACIF | | | |

| EBNA ACIF | | | |
|-----------|----|-----|-------|
| | + | - | Total |
| Biotest + | 76 | 6 | 82 |
| Biotest - | 2 | 116 | 118 |
| Total | 78 | 122 | 200 |

.............................................................................................................................................................................. Relative Sensitivity = 97.4% (C.I. = 91 to 99.7%) Relative Specificity = 95.1% (C.I. = 89.2 to 98.2%) Relative Agreement = 96.0%

#### Table 3

# Combined Site Results Direct Comparison to ACIF

| EBNA ACIF | | | |
|-----------|-----|-----|-------|
| | + | - | Total |
| Biotest + | 171 | 6 | 177 |
| Biotest - | 26 | 205 | 231 |
| Total | 197 | 211 | 408 |

Relative Sensitivity = 86.8% Relative Specificity = 97.1% Relative Agreement = 92.1%

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(C.I. = 82.1 to 91.5%) (C.I. = 93.9 to 98.9%)

Twenty-four (24) of the 25 Biotest EBNA IgG negative/ACIF positive samples were further tested with a commercially available EBNA-1 IgG ELISA method and an in-house validated Western Blot for EBNA-2. The results of this testing showed that all 24 samples were negative with the commercially available EBNA-1 IgG ELISA. Further, 21 of the 24 samples were positive in the Western Blot for EBNA-2 antibodies, indicating possible mixed reactions in the EBNA ACIF.

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Results based on Clinical Interpretation of all patient samples where Biotest ELISA EBNA-1 antibody responses matched expected serological pattern analysis for each state of infection, including - Acute, ' ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Convalescent (Late Acute or Recent Past), Past, Reactivation and Probable Reactivation/past, and Negative.

| Clinical Interpretation | | | | | |
|--------------------------------------------|-----------------------------|-----------------------------|-----------------------------|-----------------------------|----------------------------|
| | Acute | Convalescent | Past | Reactivation | Negative |
| Result matches<br>Biotest | 139 | 44 | 118 | 32 | 1 |
| Result does not<br>match Biotest | 0 | 2 | 2 | 1 | 69 |
| Sensitivity<br>Specificity<br>95% to C.I.% | 100.0%<br>N/A<br>97.4 - 100 | 95.6%<br>N/A<br>85.2 - 99.5 | 98.3%<br>N/A<br>94.1 - 99.8 | 96.9%<br>N/A<br>84.2 - 99.9 | N/A<br>98.6%<br>92.3 - 100 |

Total Clinical Sensitivity = 98.5% Total Clinical Specificity = 98.6% Total Clinical Agreement = 98.5% (C.I. = 96.6 to 99.5%) (C.I. = 92.3 to 100%)

Note: C.I. = 95% confidence intervals calculated by the exact method.

# Cross Reactivity

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No cross reactivity was observed when the Biotest EBNA IgG was used to test the following samples with detectable levels of IgG to:

| Herpes Simplex Virus I/II | (n = 27) |
|---------------------------|----------|
| Varicella Zoster Virus | (n = 50) |
| Cytomegalovirus | (n = 16) |

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# Reproducibility

To evaluate the reproducibility of the Biotest EBNA IgG ELISA, a panel of 10 patient serum specimens (low to high positive) was tested at the clinical sites. The mean, standard deviation (S.D.) -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------and coefficient of variation (C.V.) for inter-run, intra-run and inter-lab reproducibility are presented below.

| | Inter-Run (n = 10) | | | Intra-Run (n = 8) | | |
|---------|--------------------|-------|--------|-------------------|-------|--------|
| Panel # | Mean | S.D. | % C.V. | Mean | S.D. | % C.V. |
| 1 | 1.598 | 0.163 | 10.2 | 1.315 | 0.153 | 11.7 |
| 2 | 0.624 | 0.072 | 11.6 | 0.646 | 0.083 | 12.8 |
| 3 | 0.942 | 0.119 | 12.6 | 0.642 | 0.083 | 12.9 |
| 4 | 0.306 | 0.042 | 13.7 | 0.244 | 0.022 | 8.8 |
| 5 | 1.548 | 0.120 | 7.7 | 1.124 | 0.173 | 15.4 |
| 6 | 0.583 | 0.087 | 14.9 | 0.418 | 0.021 | 4.9 |
| 7 | 0.722 | 0.102 | 14.1 | 0.571 | 0.060 | 10.5 |
| 8 | 1.836 | 0.303 | 16.5 | 1.808 | 0.123 | 6.8 |
| 9 | 1.927 | 0.187 | 9.7 | 2.032 | 0.283 | 13.9 |
| 10 | 2.408 | 0.262 | 10.9 | 2.153 | 0.104 | 4.8 |

Site #1

Site #2

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| | Inter-Run (n = 4) | | | Intra-Run (n = 24) | | |
|---------|-------------------|-------|--------|--------------------|-------|--------|
| Panel # | Mean | S.D. | % C.V. | Mean | S.D. | % C.V. |
| 1 | 1.504 | 0.167 | 11.1 | 1.314 | 0.212 | 16.2 |
| 2 | 0.801 | 0.134 | 16.7 | 0.609 | 0.109 | 17.9 |
| 3 | 1.208 | 0.165 | 13.7 | 0.964 | 0.161 | 16.7 |
| 4 | 0.348 | 0.047 | 13.6 | 0.423 | 0.082 | 19.4 |
| 5 | 1.478 | 0.085 | 5.8 | 1.412 | 0.269 | 19.0 |
| 6 | 0.635 | 0.121 | 19.1 | 0.683 | 0.112 | 16.3 |
| 7 | 0.706 | 0.119 | 16.8 | 0.740 | 0.127 | 17.2 |
| 8 | 1.949 | 0.212 | 10.9 | 2.181 | 0.108 | 5.0 |
| 9 | 2.280 | 0.264 | 11.6 | 2.335 | 0.233 | 10.0 |
| 10 | 2.908 | 0.343 | 11.8 | 2.676 | 0.290 | 10.9 |

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| Inter-Lab (n = 14) | | |
|--------------------|--|--|
|--------------------|--|--|

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1 14

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| Panel # | Mean | S.D. | % C.V. |
|---------|-------|-------|--------|
| 1 | 1.571 | 0.164 | 10.4 |
| 2 | 0.674 | 0.121 | 17.9 |
| 3 | 1.018 | 0.178 | 17.5 |
| 4 | 0.318 | 0.046 | 14.5 |
| 5 | 1.528 | 0.113 | 7.4 |
| 6 | 0.598 | 0.096 | 16.0 |
| 7 | 0.718 | 0.102 | 14.3 |
| 8 | 1.868 | 0.277 | 14.8 |
| 9 | 2.028 | 0.260 | 12.8 |
| 10 | 2.551 | 0.360 | 14.1 |

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Public Health Service

Image /page/9/Picture/2 description: The image is a black and white logo for the U.S. Department of Health & Human Services. The logo consists of a stylized eagle with three stripes forming its body and wings. The eagle is enclosed in a circle with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" surrounding it. The text is in all caps and is arranged in a circular fashion to match the shape of the logo.

Food and Drug Administration 9200 Corporate Boulevard Rockville MD 20850

JUN 0 1999

Ms. Patricia E. Bonness Official Correspondent Biotest Diagnostics Corporation 66 Ford Road Suite 131 Denville, New Jersey 07834

Re:

K983841 Trade Name: Biotest Anti-EBV Recombinant EBNA IgG Regulatory Class: I Product Code: LSE Dated: March 15, 1999 Received: March 31, 1999

Dear Ms. Bonness:

We have reviewed your Section 510(k) notification of intent to market the device referenced above and we have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.

If your device is classified (see above) into either class II (Special Controls) or class III (Premarket Approval), it may be subject to such additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 895. A substantially equivalent determination assumes compliance with the Current Good Manufacturing Practice requirements, as set forth in the Quality System Regulation (QS) for Medical Devices: General regulation (21 CFR Part 820) and that, through periodic QS inspections, the Food and Drug Administration (FDA) will verify such assumptions. Failure to comply with the GMP regulation may result in regulatory action. In addition, FDA may publish further announcements concerning your device in the Federal Register. Please note: this response to your premarket notification submission does not affect any obligation you might have under sections 531 through 542 of the Act for devices under the Electronic Product Radiation Control provisions, or other Federal laws or regulations.

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Page 2

Under the Clinical Laboratory Improvement Amendments of 1988 (CLIA-88), this device may require a CLIA complexity categorization. To determine if it does, you should contact the Centers for Disease Control and Prevention (CDC) at [phone].

This letter will allow you to begin marketing your device as described in your 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801 and additionally 809.10 for in vitro diagnostic devices), please contact the Office of Compliance at [phone]. Additionally, for questions on the promotion and advertising of your device, please contact the Office of Compliance at [phone]. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 807.97). Other general information on your responsibilities under the Act may be obtained from the Division of Small Manufacturers Assistance at its toll free number [phone] or at [phone] or at its internet address "http://www.fda.gov/cdrh/dsmamain.html"

Sincerely yours,

Steven Sutman

Steven I. Gutman, M.D., M.B.A. Director Division of Clinical Laboratory Devices Office of Device Evaluation Center for Devices and Radiological Health

Enclosure

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510(k) Number (if known): ____________________________________________________________________________________________________________________________________________________

Device Name:__________________________________________________________________________________________________________________________________________________________________

Indications For Use:

The Biotest EBNA IgG ELISA is an enzyme immunoassay for the detection of IgG antibodies to the Epstein-Barr (EBV) EBNA-1 (Nuclear Antigen-1) in human serum or plasma.

It is indicated for use, in conjunction with other clinical and patient data obtained in assays for other Epstein-Barr antigens such as Early Antigen IgG and IgM, in the serological diagnosis of EBV infection.

# (PLEASE DO NOT WRITE BELOW THIS LINE-CONTINUE ON ANOTHER PAGE IF NEEDED)

______________________________________________________________________________________________________________________________________________________________________________

Concurrence of CDRH, Office of Device Evaluation (ODE)

| | Woody Debois |
|---------------|-----------------------------------------|
| | (Division Sign-off) |
| | Division of Clinical Laboratory Devices |
| 510(k) Number | K983841 |

| Prescription Use | <div style="display:inline-block;">X</div> | OR | Over-The-Counter Use ______ |
|----------------------|--------------------------------------------|----|-----------------------------|
| (Per 21 CFR 801.109) | | | (Optional Format 1-2-96) |