(222 days)
The Biotest EA IgG ELISA is an enzyme immunoassay using recombinant antigens for the qualitative detection of IgG 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 IgM and anti-EBNA-1 IgG, assist in serological diagnosis of EBV infection in pediatric and adult populations.
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 IgG or IgM antibodies are labeled. This is performed by addition of murine monoclonal anti-human IgG or 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.
The Biotest EA IgG ELISA is an enzyme immunoassay designed for the qualitative detection of IgG antibodies to Epstein-Barr Virus Early Antigens (EA) p54 and p138 in human serum or plasma. It is intended for use in conjunction with other clinical and patient data from assays for other EBV-specific antibodies (e.g., anti-Early Antigen IgM and anti-EBNA-1 IgG) to aid in the serological diagnosis of EBV infection.
Here's an analysis of the acceptance criteria and the study that proves the device meets these criteria:
1. Table of Acceptance Criteria and Reported Device Performance
The provided document does not explicitly state pre-defined acceptance criteria in terms of specific numeric thresholds for sensitivity, specificity, or agreement. Instead, it presents the results of comparative studies and implicitly uses the performance of existing methods as a benchmark for substantial equivalence. For the purpose of this analysis, we will infer the "acceptance criteria" as the performance demonstrated to achieve substantial equivalence.
| Acceptance Criteria (Implied) | Reported Device Performance (Biotest EA IgG) |
|---|---|
| Relative Sensitivity (vs. IFA) | ≥ 78.4% (Combined Sites) |
| Relative Specificity (vs. IFA) | ≥ 97.3% (Combined Sites) |
| Relative Agreement (vs. IFA) | ≥ 83.6% (Combined Sites) |
| Clinical Sensitivity (vs. Int.) | 98.5% |
| Clinical Specificity (vs. Int.) | 100% |
| Clinical Agreement (vs. Int.) | 98.7% |
| No cross-reactivity with other viruses | Observed |
| No cross-reactivity with autoantibodies | Observed |
| Inter-run %CV (Reproducibility) | Max of 16.6% (Site 1), 17.4% (Site 2) |
| Intra-run %CV (Reproducibility) | Max of 13.2% (Site 1), 19.2% (Site 2) |
| Inter-lab %CV (Reproducibility) | Max of 19.8% |
Note: "vs. IFA" refers to direct comparison with commercially available or published EA IgG IFA tests. "vs. Int." refers to comparison with clinical interpretation based on antibody patterns and clinical diagnosis.
2. Sample Size Used for the Test Set and Data Provenance
- Sample Size for Test Set: 408 patient samples.
- Data Provenance: The samples were obtained from both pediatric and adult patients (ages 1 to 74) at two geographically distinct locations. The samples represented various stages of EBV infection: acute (139), late acute (34), recent past (12), past (120), reactivation (22), past/probable reactivation (11), and negative (70). The study was a clinical study, suggesting it was prospective in nature, collecting and testing samples for a specific evaluation. The country of origin is not explicitly stated, but the submission is to the US FDA, implying that the study was likely conducted in the US or under US regulatory standards.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications
The document does not explicitly state the number of experts used or their specific qualifications (e.g., "radiologist with 10 years of experience"). However, it mentions two methods for evaluating performance:
- Direct comparison with IFA: This implies the "ground truth" for this comparison was the results from commercially available or published EA IgG IFA tests. The expertise would lie within the validated methodology and interpretation of those IFA tests.
- Comparison with clinical interpretation: This "ground truth" was based on "serological pattern analysis for each state of infection, including acute, convalescent, Past, Reactivation and Probable Reactivation/past, and Negative." This process would inherently involve expert medical knowledge and clinical judgment (likely from infectious disease specialists or clinical microbiologists) to establish the "stage of infection based on antibody patterns and clinical diagnosis at the time the specimen was drawn."
4. Adjudication Method for the Test Set
The document does not describe an explicit adjudication method (e.g., 2+1, 3+1). For the direct comparison with IFA, it's a direct comparison of results. For the comparison with clinical interpretation, the ground truth was derived from "expected serological pattern analysis" and "clinical diagnosis," which implies a consensus or established criteria for determining the infection stage, rather than individual expert adjudication of each case.
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, and the Effect Size of How Much Human Readers Improve with AI vs. Without AI Assistance
This section is not applicable to the provided document. The Biotest EA IgG ELISA is an in vitro diagnostic (IVD) device, specifically an immunoassay for detecting antibodies, not an AI-powered diagnostic imaging or decision support tool that assists human readers. Therefore, an MRMC comparative effectiveness study involving AI assistance for human readers was not performed or described.
6. If a Standalone (i.e., Algorithm Only Without Human-in-the-Loop Performance) Was Done
This section is not applicable in the context of an immunoassay. The Biotest EA IgG ELISA is itself a standalone diagnostic test. Its performance is evaluated intrinsically through its ability to detect antibodies in patient samples, not as an algorithm separate from human intervention for interpretation in the same way "standalone algorithm performance" is typically discussed for imaging AI. The "algorithm" here is the chemical and enzymatic reaction within the ELISA, and its output (spectrophotometer reading) is directly interpreted.
7. The Type of Ground Truth Used
Two types of ground truth were used for different aspects of performance evaluation:
- Established Diagnostic Tests (IFA): For the relative sensitivity and specificity, the ground truth was derived from the results of commercially available or published EA IgG Immunofluorescence Assay (IFA) tests.
- Expert Clinical Interpretation/Serological Pattern Analysis: For clinical sensitivity and specificity, the ground truth was established by "clinical interpretation (stage of infection based on antibody patterns and clinical diagnosis at the time the specimen was drawn)." This is a form of expert consensus or established diagnostic criteria based on known serological markers and patient presentation.
8. The Sample Size for the Training Set
The document does not explicitly mention a "training set" in the context of machine learning. The device is an ELISA kit, which is a biochemical assay, not an algorithm that requires a separate training phase with a distinct dataset. The "development" of the highly purified EBV antigens (EBNA-1 p72, EA-D p64, EA p138) and the optimization of the ELISA system would have involved internal validation and optimization studies by Biotest, but these are not referred to as a "training set" in the sense of AI model development.
9. How the Ground Truth for the Training Set Was Established
As noted in point 8, there is no explicit mention of a "training set" as it would be understood in machine learning. The "ground truth" in the development of an immunoassay would be established through:
- Antigen purity and specificity: Ensuring the recombinant antigens (p72, p54, p138) correctly represent the target EBV EA epitopes without undesirable cross-reactivity. This would involve molecular biology techniques, protein characterization, and immunochemical assays.
- Assay optimization: Fine-tuning reagent concentrations, incubation times, and washing steps to achieve optimal signal-to-noise ratio, linearity, and precision using well-characterized positive and negative control samples. These controls would be independently verified (e.g., by reference methods or clinical status).
§ 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).