The ImmunoProbe anti-EBNA-1 IgG assay may be used in conjunction with other Epstein-Barr serologies (VCA IgG, VCA IgM, EA (R&D), and heterophile) as an aid in the diagnosis of infectious mononucleosis. For In Vitro Diagnostic Use Only.

Device Description

The Epstein-Barr Virus Nuclear Antigen 1 (EBNA-1) IgG kit is an Enzyme-Linked Immunosorbent Assays (ELISA) for the semi-quantitative determination of IgG antibodies in human serum to EBNA-1 antigen. The EBNA-1 IgG EIA test is an enzyme linked immunosorbent assay to detect IgG antibodies to EBNA-1. Purified recombinant EBNA-1 is attached to a solid phase microtiter well. Diluted test sera is added to each well. If the antibodies are present that recognize the antigen, they will bind to the antigen in the well. After incubation the wells are washed to remove unbound antibody. An enzyme labeled anti-human IgG is added to each well. If antibody is present it will bind to the antibody attached to the antigen on the well. After incubation the wells are washed to remove unbound conjugate. A substrate solution is added to each well. If enzyme is present the substrate will undergo a color change. After an incubation period the reaction is stopped and the color intensity is measured photometrically, producing an indirect measurement of specific antibody in the patient specimen.

AI/ML Overview

This document describes the performance characteristics of the Immuno Probe EBNA-1 IgG EIA Test Kit (K951549) and presents studies to demonstrate its safety and effectiveness.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria for the EBNA-1 IgG EIA Test Kit are not explicitly stated as distinct, pre-defined thresholds in the provided text. Instead, the document presents performance characteristics and demonstrates that the device's results are in line with a predicate device and expected clinical serological profiles for EBNA-1 IgG. Based on the "Performance Characteristics" section, we can infer the following performance metrics and observed results:

Performance Metric	Acceptance Criteria (Inferred)	Reported Device Performance
Relative Sensitivity (vs. predicate)	High sensitivity (>90%)	97.8% (95% CI: 96.1% - 99.6%)
Relative Specificity (vs. predicate)	High specificity (>90%)	100% (95% CI: 96.0% - 100%)
Relative Agreement (vs. predicate)	High agreement (>90%)	98.3% (95% CI: 96.9% - 99.7%)
Inter-site Precision (CV%)	Low variability (<15-20% for clinical assays)	High Positive: 7.70%Mid Positive: 7.92%Low Positive: 10.78%Calibrator: 2.71%High Positive Control: 6.07%
Linearity (r value)	High correlation (>0.95 typically)	0.97 - 0.98
Cross-Reactivity	No cross-reactivity	No cross-reactivity observed with HSV I & II, CMV, and VZV
Sensitivity for 4-fold antibody rise	100% detection	100% for instances where acute sera had a value <3.70
Sensitivity (vs. serum characterization)	High sensitivity (>90%)	93.7% (95% CI: 88.7%-98.7%) and 96.4% (95% CI: 91.3%-100%) as reported for different subsets of seropositive samples
Specificity (vs. serum characterization)	High specificity (>90%)	Inferred from agreement in acute/seronegative groups
Accuracy/Agreement (vs. serum characterization)	High agreement (>90%)	94.7% (95% CI: 91.0%-98.3%)

2. Sample size used for the test set and the data provenance

Relative sensitivity and specificity (vs. predicate):
- Sample Size: 350 total specimens (276 positive, 74 negative). 8 equivocal results were excluded.
- Data Provenance: The study was conducted at three different sites: two R&D laboratories at commercial companies (Maryland and New York) and one large commercial lab (Pennsylvania). The data is retrospective, as it compares the new device's performance against an "alternate commercially available EIA assay" implying existing specimens.
Precision:
- Sample Size: For each of the four sera (High Positive, Mid Positive, Low Positive, Negative) and two controls (Calibrator, High Positive Control), samples were tested 3 times, twice a day for 20 days at 3 sites.
  - This totals to 3 * 2 * 20 * 3 = 360 individual assay points for each serum type across all sites (though the table states n=360 for "Inter Site Precision", implying this is the total number of data points aggregated for the CV calculation, not necessarily 360 distinct patient samples).
  - Calibrator: n=240
  - High Positive Control: n=120
- Data Provenance: Three different sites (locations not specified beyond "different sites"). Likely prospective as these were controlled runs of known sera/controls.
Linearity:
- Sample Size: 5 different sera were serially diluted and tested.
- Data Provenance: Not specified, but likely laboratory-generated data as it involves controlled serial dilutions.
Cross-Reactivity:
- Sample Size: 5 sera known to contain antibodies to HSV I & II, CMV, and VZV.
- Data Provenance: Not specified, but likely laboratory-generated data from specific known-positive samples.
Evaluation of paired sera:
- Sample Size: 20 high positive sera were serially diluted to create 68 paired samples with a four-fold dilution.
- Data Provenance: Not specified, but likely laboratory-generated from existing high-positive samples.
Sensitivity and Specificity Based on Serum Characterization:
- Sample Size: 150 specimens (95 seropositive, 24 acute, 31 seronegative). 8 equivocal results were excluded.
- Data Provenance: The serum was from "the first study site" which implies one of the sites mentioned in the relative sensitivity/specificity study. The characterization (seronegative, acute, seropositive) suggests these were clinical samples. The study is likely retrospective, using samples with pre-established serological profiles.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts

The document does not explicitly state the number or qualifications of experts used to establish the ground truth.

For Relative Sensitivity/Specificity: The ground truth for the comparison was an "alternate commercially available EIA assay." It is implied that the results from this predicate device are considered the truth for this comparison. The "experts" would implicitly be the developers and users of this predicate device, whose qualifications are not detailed.
For Sensitivity/Specificity Based on Serum Characterization: The ground truth was established by classifying sera as "seronegative," "acute," or "seropositive" based on "other Epstein-Barr serologies (VCA IgG, VCA IgM)." This implies a determination made by laboratory personnel or serologists who follow established diagnostic protocols. No specific number or qualifications are provided.

4. Adjudication method for the test set

The document does not describe an adjudication method involving multiple readers for the test sets. The results appear to be based on direct laboratory measurements and comparisons to a predicate device or established serological profiles. "Equivocals were not included in the calculations" for the relative sensitivity/specificity and the serum characterization studies, which acts as a form of exclusion rather than an adjudication process to assign true status.

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 performed. This device is an Enzyme-Linked Immunosorbent Assay (ELISA), which is an in vitro diagnostic test, not an AI-powered diagnostic imaging or interpretation tool that would typically involve human readers. Therefore, the concept of human readers improving with or without AI assistance is not applicable here.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done

Yes, the studies presented are all standalone performance evaluations of the assay itself. The results (e.g., optical density readings converted to index values) are generated by the kit components and detected instrumentally, without a human-in-the-loop interpretation step that influences the primary measurement outcome beyond standard laboratory procedures for running an ELISA.

7. The type of ground truth used

For Relative Sensitivity and Specificity: The ground truth was based on the results of a "commercially available EIA assay" (predicate device).
For Sensitivity and Specificity Based on Serum Characterization: The ground truth was based on serological characterization using other established EBV serologies (VCA IgG, VCA IgM) to classify samples as seronegative, acute, or seropositive. This falls under the category of established diagnostic criteria.
For Precision, Linearity, Cross-Reactivity, Paired Sera: The ground truth was based on known characteristics of the samples used (e.g., known positive/negative samples for precision, serially diluted samples for linearity, samples with known viral antibodies for cross-reactivity).

8. The sample size for the training set

The document does not explicitly mention a "training set" in the context of machine learning or algorithm development. The device is a traditional immunoassay kit, not an AI/ML algorithm. Therefore, no specific training set data is described for this type of device. The extensive testing described in the document serves as performance validation.

9. How the ground truth for the training set was established

As there is no AI/ML algorithm and thus no explicit "training set" described, this question is not applicable. The performance characteristics were established by testing against established serological methods and clinical classifications, which serve as the reference standard or "ground truth" for validation.

Summary

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APR 2 9 1996

Summary of Safety and Effectiveness Information EBNA-1 IgG EIA Test Kit

K951549

Immuno Probe Inc. I. 1306 Bailes Lane, Suite F Frederick, Maryland 21701 Contact person: William Boteler Telephone: 301-695-7920 Date of preparation: March 28, 1995

II. Description of Device

The Epstein-Barr Virus Nuclear Antigen 1 (EBNA-1) IgG kit is an Enzyme-Linked Immunosorbent Assays (ELISA) for the semi-quantitative determination of IgG antibodies in human serum to EBNA-1 antigen. The ImmunoProbe anti-EBNA-1 IgG assay may be used in conjunction with other Epstein-Barr serologies (VCA IgG, VCA IgM, EA (R&D), and heterophile) as an aid in the diagnosis of infectious mononucleosis. For In Vitro Diagnostic Use Only.

The EBNA-1 IgG EIA test is an enzyme linked immunosorbent assay to detect IgG antibodies to EBNA-1. Purified recombinant EBNA-1 is attached to a solid phase microtiter well. Diluted test sera is added to each well. If the antibodies are present that recognize the antigen, they will bind to the antigen in the well. After incubation the wells are washed to remove unbound antibody. An enzyme labeled anti-human IgG is added to each well. If antibody is present it will bind to the antibody attached to the antigen on the well. After incubation the wells are washed to remove unbound conjugate. A substrate solution is added to each well. If enzyme is present the substrate will undergo a color change. After an incubation period the reaction is stopped and the color intensity is measured photometrically, producing an indirect measurement of specific antibody in the patient specimen.

III. Predicate Device

The EBNA-1 IgG EIA test is substantially equivalent to BioWhitttaker's EBNA STAT test. Equivalence is demonstrated by the following comparative results:

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Performance Characteristics

Relative sensitivity and specificity. Three different sites compared the EBNA-1 IgG ELA test relative to a commercially available EIA assay. Two of the sites were R&D laboratories at commercial companies located in Maryland and New York. The other site was a large commercial lab located in Pennsylvania. The results of the studies are compiled and summarized in Table 1. None of the performance characteristics were established with specimens from patients having nasopharyngeal carcinoma or Burkitt's lymphoma.

Table 1 EBNA-1 IgG Relative Sensitivity and Specificity

+ eq = 270 5 + б Alternate EBNA-1 G 0 0 l eq ELISA -0 I 74

Total

281

હરે

357

ImmunoProbe EBNA-1 G EIA

Relative Sensitivity = 270/276 = 97.8	95% Confidence interval = 96.1% - 99.6%
Relative Specificity = 74/74 = 100%	95% Confidence interval = 96.0% - 100%
Relative Agreement = 344/350 = 98.3%	95% Confidence interval = 96.9% - 99.7%

8 I

270

Equivocals were not included in the above calculations.

Total

The 95% confidence interval for relative specificity was calculated assuming one false positive The 95% confidence intervals were calculated using the normal method.

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Precision. Four different sera (High Positive, Mid Positive, Low Positive, and Negative) were assayed at three different sites to determine the precision of the assay. Each sera was tested three times each, twice a day for twenty days at each of the three study sites. The inter-site coefficient of variation (CV) for each serum is presented in table i

Table 2 Inter Site Precision n= 360

	Mean	Std Dev	CV%
High Positive	4.95	0.381	7.70%
Mid Positive	2.89	0.229	7.92%
Low Positive	1.41	0.152	10.78%
Negative	0.01	0.022	not applicable
Calibrator (n=240)	4.17	0.113	2.71%
High Positive Control (n=120)	6.90	0.419	6.07%

The data in table 3 illustrates EBNA-1 index values for serially two fold 3. Linearity. diluted sera. The index values are compared to log2 of dilution by standard linear The data indicates that the antibody can be semi-quantitated by using a single regression. serum dilution.

Table 3 EBNA-1 G Linearity

Serum #	Neat	1:2	1:4	1:8	1:16	1:32	1:64	SLOPE	Y INTERCEPT	r
1	8.6	5.96	3.50	1.96	0.87			1.95	-1.66	0.98
2	11.4	9.30	6.90	4.10	2.50	1.30	0.42	1.91	-2.49	0.98
3	6.90	4.90	2.7	1.5	0.69			1.58	-1.41	0.98
4	8.7	6.5	4.07	2.32	1.20	0.55		1.67	-1.95	0.97
5	9.4	7.17	4.8	3.2	1.5	0.70		1.77	-1.75	0.98

Linear regression compared Index Values to log2 of dilution.

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Serum containing IgG antibody detectable by ELISA to Herpes 4. Cross-Reactivity. Simplex Virus I & II, Cytomegalovirus, and Varicella Zoster Virus were assayed. The data summarized in Table 4 indicates that antibodies to these Herpes Viruses do not crossreact with the EBNA-1 IgG EIA kit.

TABLE 4

Cross Reactivity

SERUM	EBNA-1 G	HSVI G	HSV2 G	CMV	VZV
1	0.67 -	2.43 +	0.60 -	1.69 +	2.28 +
2	0.36 -	0.32 +	0.08 -	0.18 -	3.17 +
3	0.73 -	3.67 +	0.86 -	1.11 +	2.45 +
4	0.29 -	0.30 -	0.39 -	0.18 -	3.21 +
5	0.34 -	6.18 +	5.98 +	0.18 -	2.14 +

Sera ≥ 1.10 were considered positive. Sera ≤ 0.90 were considered negative.

Evaluation of paired sera. To validate the sensitivity of the paired sera procedure 20 high positive sera were serially two fold diluted and run on the assay. From these dilutions, there were 68 pairs that had a four fold dilution where the acute sera had a value of less than 3.70. All 68 pairs demonstrated a >46% rise in ISR value, showing a significant rise in antibody. Therefore the paired sera procedure demonstrated 100% sensitivity in being able to detect a four fold increase in antibody level when the acute sera has a value of <3.70.

6.Sensitivity and Specificity Based on Serum Characterization

The serum from the first study site were characterized as seronegative ( no serological evidence of past or present EBV infection), acute (VCA IgM present), or seropositive (presence of VCA IgG antibodies, no evidence of VCA IgM, indicative of past infection). The sensitivity, specificity and accuracy of the assay was determined based on this characterization. It was assumed that the EBNA-1 IgG response should be negative for seronegative and acute serum, and positive for seropositive serum. The results are summerized in Table 5.

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Table 5

		SeropositiveVCA IgG +VCA IgM -	AcuteVCA IgM+	SeronegativeVCA IgG -VCA IgM -	Total
IPIEBNA-1 IgG	Positive	89	1	1	91
	Negative	6	23	30	59
	Total	95	24	31	150
Relative Sensitivity = 89/95 = 93.7%Relative Sensitivity = 53/55 = 96.4%Relative Agreement= 142/150 = 94.7%			95% Confidence Interval = 88.7%-98.7%95% Confidence Interval = 91.3%-100%95% Confidence Interval = 91.0%-98.3%

Eight equivocal results were not included in the calculations. The 95% confidence intervals were calculated using the normal method.

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).