The Biotest EA IgM ELISA is an enzyme immunoassay using recombinant antigens for the qualitative detection of IgM antibodies to the Epstein-Barr Virus early antigens (EA) p54 and p138 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 virus-specific antibodies such as anti-Early Antigen IgG and anti-EBNA-1 IgG, assist in serological diagnosis of EBV infection in pediatric and adult populations.

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).

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 IgM antibodies are labeled. This is performed by addition of murine monoclonal anti-human IgM 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.

AI/ML Overview

This appears to be a 510(k) summary for the Biotest Anti-EBV Recombinant EA IgM ELISA test. Let's break down the acceptance criteria and study details.

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined "acceptance criteria" in terms of specific performance thresholds for sensitivity and specificity. Instead, it presents the results of a comparison study against a predicate device and clinical interpretation. However, we can infer the reported performance metrics.

Performance Metric	Acceptance Criteria (Implied)	Reported Device Performance
Primary Comparison (vs. VCA IgM/IgG IFA)	Not explicitly stated, but demonstrating substantial equivalence to the predicate device in terms of diagnostic performance.	Relative Sensitivity: 84.1% (Combined Sites) Relative Specificity: 89.4% (Combined Sites) Relative Agreement: 87.0% (Combined Sites)
Clinical Interpretation	Not explicitly stated, but demonstrating agreement with expected serological patterns.	Total Clinical Sensitivity: 98.2% Total Clinical Specificity: 97.1% Total Clinical Agreement: 98.0%
Cross-Reactivity	No unexpected cross-reactivity with common interfering substances (HSV, VZV, CMV, Toxoplasma, RF, ANA, ASL).	No cross-reactivity observed in tested rheumatoid factor positive, antinuclear antibody positive, and ASL positive samples. Limited non-reactivity in a subset of samples from patients acutely infected with HSV I/II, VZV, CMV, Toxoplasma (e.g., 4 of 5 for HSV I/II).
Reproducibility	Acceptable inter-run, intra-run, and inter-lab variability.	Inter-run % C.V.: 9.7% - 16.9% (Site 1), 3.1% - 7.7% (Site 2) Intra-run % C.V.: 2.8% - 6.3% (Site 1), 6.8% - 14.7% (Site 2) Inter-lab % C.V.: 7.9% - 15.7%

2. Sample Size Used for the Test Set and Data Provenance

Sample Size for Test Set: 408 patient samples.
Data Provenance: Clinical study conducted at two geographically distinct locations. Samples were obtained from both pediatric and adult patients (ages 1 to 74). The study is retrospective in the sense that samples were collected and categorized prior to testing with the Biotest ELISA, allowing for comparison to existing diagnostic methods and clinical records. The document doesn't specify countries of origin, but given the US FDA submission, it's highly likely to be within the US.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts

The document does not explicitly detail the number or qualifications of experts used to establish the "ground truth" for the test set.

For the "direct comparison with VCA IgM/IgG IFA", the predicate device (Gull VCA IGM test kits, which is an Indirect Fluorescent Antibody (IFA) assay) serves as the reference, which itself would have been interpreted by trained laboratory personnel.
For the "comparison with clinical interpretation," the ground truth was "based on antibody patterns and clinical diagnosis at the time the specimen was drawn." This implies that the initial diagnoses and serological classifications (e.g., acute, past, negative) were made by healthcare professionals and laboratory personnel, but no specific number or qualification of these experts is provided in this summary.

4. Adjudication Method for the Test Set

The document does not explicitly describe an adjudication method for conflicting results.

For the direct comparison, it appears the results of the Biotest EA IgM ELISA were compared directly to the results of the VCA IgM/IgG IFA, with agreement or disagreement noted.
For the clinical interpretation, "Result matches Biotest" and "Result does not match Biotest" categories suggest a direct comparison of the Biotest result against an established clinical interpretation for each sample.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, If So, What Was the Effect Size of How Much Human Readers Improve with AI vs Without AI Assistance

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. This document describes the validation of an ELISA diagnostic kit, not an AI-powered image analysis or diagnostic tool that would involve human readers interpreting results with or without AI assistance. Therefore, there is no effect size for human reader improvement with AI.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done

Yes, a standalone performance evaluation was done. The Biotest EA IgM ELISA is a laboratory diagnostic test. Its performance (sensitivity, specificity, reproducibility, cross-reactivity) was evaluated as a standalone device, meaning the algorithm/assay itself directly produces the result (positive/negative) without human intervention in the interpretation of the assay's output during the performance study itself. Human intervention would be involved in performing the test and interpreting the final clinical significance, but the device's diagnostic output is standalone.

7. The Type of Ground Truth Used

Two types of ground truth were used:

Predicate Device Comparison: The results of commercially available or published EBV VCA IgM/IgG IFA tests. This serves as a "reference standard" from an established, legally marketed diagnostic method.
Clinical Interpretation: "Stage of infection based on antibody patterns and clinical diagnosis at the time the specimen was drawn." This combines serological profiles (antibodies against other EBV antigens) with the clinical presentation of the patient. This represents a more comprehensive "clinical ground truth."

8. The Sample Size for the Training Set

The document does not specify a separate "training set" or its sample size. This is typical for traditional immunoassay device submissions. The "clinical study of 408 patient samples" appears to be the primary validation set (test set) for evaluating the device's performance characteristics. Diagnostic kits like this are generally developed and optimized during an internal R&D phase, and the studies presented are for validation rather than training of a machine learning model.

9. How the Ground Truth for the Training Set Was Established

As no explicit "training set" is mentioned in the context of machine learning model development, this question is not directly applicable. For the development and optimization of the ELISA kit itself (which would be analogous to "training" in a broad R&D sense), the ground truth would have been established through extensive research using characterized EBV-infected and uninfected samples, likely verified by a combination of established serological methods, PCR, and clinical diagnosis, but these details are not part of this 510(k) summary.

Summary

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JUN g 1999

K983842

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 Biotest Diagnostics Corporation Company Name and Address: 66 Ford Road, Suite 131 Denville, NJ 07834 (609) 397-8511 Telephone: (609) 397-8224 FAX: Patricia E. Bonness, Official Correspondent Contact Person: Date 510fk) Summary was Prepared: October 26, 1998 B. Device Names Biotest Anti-EBV Recombinant Proprietary Name: EA IgM EBV EA IgM Common Name: Epstein-Barr virus serological reagents Classification Name:
C. Legally Marketed Device

Biotest Diagnostics claims substantial equivalence to the EBV IgM Test (K822734) currently in commercial distribution by Gull Laboratories, Inc., Salt Lake City, UT.

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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BiotestAnti-EBVrecombinant	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

E. Intended Use

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Comparison with Predicate Device F.
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ﻧﮯ

A summary comparison of the features of the Biotest EA IgM and the Gull VCA IGM test kits is provided in Table 1 below:

Table 1 Feature Comparison of Biotest and Gull EBV IgM Test Kits

	Biotest	Gull
Intended Use	Detection of IgM antibodiesto EBV EAQualitative only	Detection of IgM antibodiesto EBV VCAQualitative only
Assay Method	ELISA	Indirect fluorescent antibody (IFA)
Reactive Ingredients	Recombinant EBV (EA p54/p138)Peroxidase-conjugated monoclonalanti-human IgM (mouse)	HR1, Burkitt's lymphocytic cellsFluorescein-labeledanti-human IgM
Specimen: TypeMin. VolumeStorage	Serum or plasma25 µl2 - 8°C or -20°C	Serum15 µl of 1:10 and 1:40 dilutions2 - 8°C/7days or -20°C
Controls	NegativePositive	NegativePositive
Chromogen	TMB	Fluorescein
Results:Evaluation	spectrophotometer @ 450 nm	Fluorescence microscope
Kit Size	96 tests	50 or 100 tests

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G. Performance Data

Sensitivity/Specificity

The performance of the Biotest EA IgM 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 EA IgM ELISA: direct comparison with commercially available or published EA IgM IFA 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 VCA IgM/IgG IFA for both sites combined demonstrated a relative sensitivity of 84.1% and a relative specificity of 89.4%.

Table 1

Clinical Site 1 Direct Comparison to VCA IgM/IgG IFA

VCA IgM/IgG IFA
	+	-	Total
Biotest +	73	13	86
Biotest -	14	108	122
Total	87	121	208

Relative Sensitivity = 83.9% Relative Specificity = 89.3% Relative Agreement = 87.0% (C.I. = 74.5 to 90.9%) (C.I. = 83.7 to 94.8%)

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Table 2

VCA IgM/IgG IFA
	+	-	Total
Biotest +	80	11	91
Biotest -	15	94	109
Total	95	105	200

Clinical Site 2 Direct Comparison to VCA IgM/IgG IFA

《《 Relative Sensitivity = 84.2% Relative Specificity = 89.5% Relative Agreement = 87.0%

..............................................................................................................................................................................

)

(C.I. = 75.3 to 90.0%) (C.I. = 83.7 to 95.4%)

Table 3

Combined Site Results Direct Comparison to VCA IgM/IgG

VCA IgM/IgG
	+	-	Total
Biotest +	153	24	177
Biotest -	29	202	231
Total	182	226	408

Relative Sensitivity = 84.1% Relative Specificity = 89.4% Relative Agreement = 87.0%

(C.I. = 78.7 to 89.4%) (C.I. = 85.4 to 93.4%)

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Results based on Clinical Interpretation of all patient samples where Biotest ELISA EA IgM 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 matchesBiotest	138	45	117	32	2
Result does notmatch Biotest	1	1	3	1	68
SensitivitySpecificity95% to C.I.%	99.2%N/A96.1 - 100	97.8%N/A88.5 - 99.9	97.5%N/A92.9 - 99.5	96.9%N/A84.2 - 99.9	N/A97.1%90.1 - 99.7

Total Clinical Sensitivity = 98.2% Total Clinical Specificity = 97.1% Total Clinical Agreement = 98.0% (C.I. = 96.2 to 99.3%) (C.I. = 90.1 to 99.7%)

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

Cross Reactivity

No cross reactivity was observed when the Biotest EA IgM ELISA was used to test samples from patients acutely infected with:

Herpes Simplex Virus I/II	4 of 5 samples found non-reactive
Varicella Zoster Virus	5 of 11 samples found non-reactive
Cytomegalovirus	3 of 9 samples found non-reactive
Toxoplasma	4 of 5 samples found non-reactive

Further, no cross reactivity was observed in tests of rheumatoid factor positive (n = 10), antinuclear antibody positive (n = 5) and ASL positive (n = 15) samples.

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Reproducibility

ﻜﺴﺎ

To evaluate the reproducibility of the Biotest EA IgM 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 = 11)			Intra-Run (n = 8)
Panel #	Mean	S.D.	% C.V.	Mean	S.D.	% C.V.
1	1.799	0.175	9.7	1.801	0.065	3.6
2	1.013	0.171	16.9	1.140	0.032	2.8
3 -	1.280	0.183	14.3	1.286	0.070	5.5
4	0.294	0.033	11.2	0.287	0.018	6.3
5	1.065	0.119	11.2	1.189	0.059	5.0
6	0.651	0.088	13.5	0.680	0.034	5.1
7	1.231	0.152	12.3	1.325	0.060	4.5
8	0.959	0.130	13.5	0.946	0.060	6.3
9	0.798	0.100	12.5	0.811	0.037	4.6
10	0.664	0.067	10.0	0.711	0.020	2.9

Site #1

Site #2

	Inter-Run (n = 5)			Intra-Run (n = 24)
Panel #	Mean	S.D.	% C.V.	Mean	S.D.	% C.V.
1	1.641	0.127	7.7	1.829	0.268	14.7
2	1.165	0.036	3.1	1.257	0.164	13.1
3	1.415	0.043	3.1	1.471	0.124	8.4
4	0.335	0.020	6.0	0.362	0.052	14.4
5	1.271	0.063	5.0	1.372	0.127	9.3
6	0.664	0.032	4.8	0.773	0.076	9.9
7	1.360	0.083	6.1	1.522	0.104	6.8
8	1.009	0.075	7.4	1.111	0.099	8.9
9	0.932	0.037	3.9	0.997	0.070	7.0
10	0.705	0.019	2.7	0.783	0.048	6.2

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Inter-Lab (n = 14)
Panel #	Mean	S.D.	% C.V.
1	1.75	0.172	9.8
2	1.061	0.167	15.7
3	1.322	0.173	13.1
4	0.307	0.037	11.9
5	1.129	0.140	12.4
6	0.655	0.077	11.8

1.271

0.974

0.840

0.677

12.0

12.1

13.1

7.9

0.153

0.117

0.110

0.053

Inter-Lab (n = 14)

)

7 *

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Image /page/9/Picture/1 description: The image shows the logo for the U.S. Department of Health and Human Services. The logo consists of a circular seal with the words "DEPARTMENT OF HEALTH & HUMAN SERVICES • USA" arranged around the perimeter. Inside the circle is an abstract symbol that resembles an eagle or bird-like figure with three stylized lines forming its body and wings.

Food and Drug Administration 9200 Corporate Boulevard Rockville MD 20850

JUN 9 1999

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

Re: K983842

Trade Name: Biotest Anti-EBV Recombinant EA IgM 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 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 (770)488-7655.

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 (301) 594-4588. Additionally, for questions on the promotion and advertising of your device, please contact the Office of Compliance at (301) 594-4639. 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 (800) 638-2041 or at (301) 443-6597 or at its internet address "http://www.fda.gov/cdrh/dsmamain.html"

Sincerely yours.

Steven Autman

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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Page _ of _

J10(k) Number (if known): 4983892

Device Name: _________________________________________________________________________________________________________________________________________________________________

Indications For Use:

The Biotest EA IgM is an enzyme immunoassay for the detection of IgM antibodies to the Epstein-Barr Virus (EBV-EA (Early Antigens) p54/p138) 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 EBNA IgG and Early Antigen IgG, 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 Dubois (Division Sign Off)
	Division of Clinical Laboratory Devices
	510(k) NumberK983842	510(k) Number	K983842
510(k) Number	K983842
Prescription Use X(Per 21 CFR 801.109)	OR	Over-The-Counter Use ______(Optional Format 1-2-96)

Regulation Number and Section

§ 866.3235 Epstein-Barr virus serological reagents.

(a)
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).(b)
Classification. Class I (general controls).