Nano-Check COVID-19 Antigen Test
Device Facts
| Record ID | K231187 |
|---|---|
| Device Name | Nano-Check COVID-19 Antigen Test |
| Applicant | Nano-Ditech Corporation |
| Product Code | QVF · Microbiology |
| Decision Date | Jan 23, 2024 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3982 |
| Device Class | Class 2 |
Indications for Use
The Nano-Check COVID-19 Antigen Test is a lateral flow immunochromatographic assay for the rapid, qualitative detection of SARS-CoV-2 nucleoprotein protein antigens directly in anterior nasal swab specimens from individuals with signs and symptoms of upper respiratory infection (i.e., symptomatic) when testing is started within 4 days of symptom onset. The test is intended for use as an aid in the diagnosis of SARS-CoV-2 infections (COVID-19) in symptomatic individuals when either: tested at least twice over three days with at least 48 hours between tests; or when tested once, and negative by the Nano-Check COVID-19 Antigen Test and followed with a molecular test. The test does not differentiate between SARS-CoV or SARS-CoV-2. A negative test result is presumptive, and it is recommended these results be confirmed by a molecular SARS-CoV-2 assay. Positive results do not rule out co-infection with other bacteria or viruses and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Performance characteristics for SARS-CoV-2 were established during the 2022 SARS-CoV-2 pandemic when SARS-CoV-2 Omicron was the predominant SARS-CoV-2 variant in circulation. When other SARS-CoV-2 virus variants are emerging, performance characteristics may vary.
Device Story
Nano-Check COVID-19 Antigen Test is a lateral flow immunochromatographic assay for qualitative detection of SARS-CoV-2 nucleocapsid protein antigens. Input: anterior nasal swab specimens collected from symptomatic individuals. Operation: swab placed in reagent tube with buffer to lyse virus; sample dispensed into test cassette sample well; capillary action moves sample across nitrocellulose membrane. Test strip contains biotinylated monoclonal capture antibodies and colloidal gold-labeled detection antibodies. If antigen is present, immunocomplex forms and binds to streptavidin at test line, creating visible pinkish-red line. Procedural control line confirms adequate flow. Results read visually by healthcare provider at 15-20 minutes. Output: qualitative positive, negative, or invalid result. Used in point-of-care settings by trained operators. Benefits: rapid identification of SARS-CoV-2 infection to aid clinical decision-making; serial testing protocol mitigates risk of false negatives.
Clinical Evidence
Prospective multicenter clinical study (n=670) conducted 2022-2023 during Omicron predominance. Subjects were symptomatic. Comparator: FDA-cleared RT-PCR. Results: PPA 83.67% (95% CI: 76.86-88.78%); NPA 99.62% (95% CI: 98.62-99.90%).
Technological Characteristics
Lateral flow immunochromatographic assay. Materials: nitrocellulose membrane, sample pads, biotinylated monoclonal antibodies, colloidal gold-labeled monoclonal antibodies, streptavidin. Format: test cassette. Energy source: none (capillary action). Connectivity: none. Sterilization: N/A.
Indications for Use
Indicated for symptomatic individuals with signs of upper respiratory infection within 4 days of symptom onset. For use as an aid in SARS-CoV-2 diagnosis via serial testing (at least twice over 3 days, 48 hours apart) or single test followed by molecular confirmation.
Regulatory Classification
Identification
The Sofia 2 SARS Antigen+ FIA is a lateral flow immunofluorescent sandwich assay used with the Sofia 2 instrument for the rapid, qualitative detection of SARS-CoV-2 nucleocapsid protein antigens directly in anterior nasal swab specimens from symptomatic individuals. It is intended as an aid in the diagnosis of SARS-CoV-2 infections (COVID-19) in symptomatic individuals when tested at least twice over three days with at least 48 hours between tests.
Predicate Devices
- Sofia 2 SARS Antigen+ FIA, Sofia 2 SARS Antigen+ FIA Control Swab Set (DEN220039)
Related Devices
- K232377 — Healgen Rapid COVID-19 Antigen Test · Healgen Scientific,, LLC · Apr 19, 2024
- K240728 — CorDx Tyfast COVID-19 Ag Rapid Test; CorDx COVID-19 Ag Test · CorDx, Inc. · Jun 21, 2024
- K241317 — Wondfo 2019-nCoV Antigen Test (Lateral Flow Method) · Guangzhou Wondfo Biotech Co., Ltd. · Sep 30, 2024
- K251595 — COVID-19 Detect Rapid Self -Test · InBios International, Inc. · Oct 12, 2025
Submission Summary (Full Text)
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FDA
U.S. FOOD & DRUG
ADMINISTRATION
# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
ASSAY ONLY
## I Background Information:
A 510(k) Number
K231187
B Applicant
Nano-Ditech Corporation
C Proprietary and Established Names
Nano-Check COVID-19 Antigen Test
D Regulatory Information
| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| QVF | Class II | 21 CFR 21 CFR 866.3982
- Simple Point-Of-Care Device to Directly Detect Sars-Cov-2 Viral Target From Clinical Specimens In Near-Patient Setting | |
## II Submission/Device Overview:
A Purpose for Submission:
To obtain a substantial equivalence determination for the Nano-Check COVID-19 Antigen Test
B Measurand:
Nucleocapsid protein antigen from SARS-Coronavirus 2 (SARS-CoV-2)
C Type of Test:
Qualitative lateral flow immunoassay
Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov
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III Intended Use/Indications for Use:
A Intended Use(s):
See Indications for Use below.
B Indication(s) for Use:
The Nano-Check COVID-19 Antigen Test is a lateral flow immunochromatographic assay for the rapid, qualitative detection of SARS-CoV-2 nucleoprotein protein antigens directly in anterior nasal swab specimens from individuals with signs and symptoms of upper respiratory infection (i.e., symptomatic) when testing is started within 4 days of symptom onset. The test is intended for use as an aid in the diagnosis of SARS-CoV-2 infections (COVID-19) in symptomatic individuals when either: tested at least twice over three days with at least 48 hours between tests; or when tested once, and negative by the Nano-Check COVID-19 Antigen Test and followed with a molecular test.
The test does not differentiate between SARS-CoV or SARS-CoV-2.
A negative test result is presumptive, and it is recommended these results be confirmed by a molecular SARS-CoV-2 assay.
Positive results do not rule out co-infection with other bacteria or viruses and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.
Performance characteristics for SARS-CoV-2 were established during the 2022 SARS-CoV-2 pandemic when SARS-CoV-2 Omicron was the predominant SARS-CoV-2 variant in circulation. When other SARS-CoV-2 virus variants are emerging, performance characteristics may vary.
C Special Conditions for Use Statement(s):
Rx - For Prescription Use Only
IVD - For In Vitro Diagnostic Use Only
D Special Instrument Requirements:
N/A
IV Device/System Characteristics:
A Device Description:
The Nano-Check COVID-19 Antigen Test is an immunochromatographic lateral flow assay for detection of SARS-CoV-2 nucleoprotein antigens in human anterior nasal swab specimens without transport media from those who are suspected of COVID-19 within the first 5 days of symptom onset.
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To initiate testing, a flocked swab is used to collect anterior nasal swab specimens from both nostrils. The patient sample is placed in the Reagent Tube, which is either provided pre-filled with buffer, or that the user pre-filled with buffer provided in the test kit's ampule. The buffer disrupts the virus particles in the sample, exposing internal viral nucleoproteins. After disruption, the sample is dispensed into the Test Cassette sample well.
The Test Cassette is comprised of a plastic cassette with a nitrocellulose membrane and sample pads. Specifically, the test strip contains: (1) the Sample Pad, which receives the buffer and distributes the sample across the test strip; (2) the Biotin Pad, which contains biotinylated monoclonal antibodies specific to the SARS-CoV-2 nucleocapsid antigen; (3) the Dye Pad, which contains colloidal gold particles coupled with monoclonal antibodies specific to the SARS-CoV-2 nucleocapsid antigen; (4) the Test Line, which contains embedded streptavidin to capture the antibody-antigen immunocomplexes; and (5) the Absorbent Pad, which absorbs sample after it has migrated across the nitrocellulose membrane.
The sample will migrate up the test strip via capillary action. If SARS-CoV-2 nucleoprotein antigens are present, they will bind to the biotinylated monoclonal capture antibodies present on the biotin pad. As the sample passes through the dye pad, SARS-CoV-2 nucleoprotein antigen/biotin-antibody complexes will bind to specific monoclonal detection antibodies labelled with colloidal gold particles to form an immunocomplex with the biotinylated antibody, the nucleoprotein antigen, and the colloidal gold labelled antibody. At the test line, the immunocomplexes will be captured through interaction of the biotinylated antibody of the complexes with the streptavidin embedded into the test line, concentrating the colloidal-gold labeled antigen at the test line. This will form a visible pinkish-red line. Sample continues to flow through the test device which also contains a procedural control line to assess for sample presence and adequate sample flow. A visible pinkish-red line at the control region should always appear if the assay is performed correctly to verify proper liquid flow and gold conjugation. If no visible signal appears on C line, the test result is invalid, and this sample should be tested again with another test cassette. Test results are read between 15 and 20 minutes.
External positive control and negative control swabs are provided with each kit of Nano-Check COVID-19 Antigen Tests and should be processed according to the IFU upon receiving a new lot of test kits. The control swabs are intended to be used as quality control samples representative of positive and negative test samples to demonstrate that the reagents are functional, and the assay procedure is performed correctly.
## B Principle of Operation:
The Nano-Check COVID-19 Antigen Test employs lateral flow technology in a sandwich design to detect nucleocapsid protein from SARS-CoV-2 in anterior nasal samples. Following sample collection, the swab is placed in the reagent tube containing extraction buffer to lyse the sample and solubilize viral nucleoproteins. After lysis, two drops of the sample are loaded into the test cassette sample well. The sample migrates across a pad containing biotinylated capture antibodies, a pad containing colloidal gold-labelled detection antibodies, and then the test strip including the test and control lines. If SARS-CoV-2 viral antigens are present, they will be captured and bound by the antibodies and the biotinylated immunocomplex will be bound to streptavidin embedded on the Test Line. The test results are visually read. A pink-red line at the Test Line and a pink-red line at the Control Line indicate a positive sample. A pink-red line at
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the Control Line only indicates a negative sample. Any test without a visible line at the Control Line should be interpreted as invalid and testing should be repeated with a new device.
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V Substantial Equivalence Information:
A Predicate Device Name(s):
Sofia 2 SARS Antigen+ FIA, Sofia 2 SARS Antigen+ FIA Control Swab Set
B Predicate 510(k) Number(s):
DEN220039
C Comparison with Predicate(s):
| Device & Predicate Device(s): | Device
K231187 | Predicate
DEN220039 |
| --- | --- | --- |
| Device Trade Name | Nano-Check COVID-19 Antigen Test | Sofia 2 SARS Antigen+ FIA, Sofia 2 SARS Antigen+ FIA Control Swab Set |
| General Device Characteristic Similarities | | |
| Intended Use/Indications For Use | The Nano-Check COVID-19 Antigen Test is a lateral flow immunochromatographic assay for the rapid, qualitative detection of SARS-CoV-2 nucleocapsid protein antigens directly in anterior nasal swab specimens from individuals with signs and symptoms of upper respiratory infection (i.e., symptomatic) when testing is started within 4 days of symptom onset. The test is intended for use as an aid in the diagnosis of SARS-CoV-2 infections (COVID-19) in symptomatic individuals when either: tested at least twice over three days with at least 48 hours between tests; or when tested once, and negative by the Nano-Check COVID-19 Antigen Test and followed up with a molecular test. The test does not differentiate between SARS-CoV and SARS-CoV-2.
A negative test result is presumptive, and it is recommended these results be confirmed by a molecular SARS-CoV-2 assay. Negative results do not preclude SARS-CoV-2 infections and should not be used as the sole basis for treatment or other patient | The Sofia 2 SARS Antigen+ FIA is a lateral flow immunofluorescent sandwich assay that is used with the Sofia 2 instrument for the rapid, qualitative detection of SARS-CoV-2 nucleocapsid protein antigens directly in anterior nasal swab specimens from individuals with signs and symptoms of upper respiratory infection (i.e., symptomatic) when testing is started within 6 days of symptom onset. The test is intended for use as an aid in the diagnosis of SARS-CoV-2 infections (COVID-19) in symptomatic individuals when tested at least twice over three days with at least 48 hours between tests. The test does not differentiate between SARS-CoV and SARS-CoV-2.
A negative test result is presumptive, and it is recommended these results be confirmed by a molecular SARS-CoV-2 assay. Negative results do not preclude SARS-CoV-2 infections and should not be used as the sole basis for treatment or other patient |
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| Device & Predicate Device(s): | Device
K231187 | Predicate
DEN220039 |
| --- | --- | --- |
| | CoV-2 assay. Positive results do not rule out co-infection with other bacteria or viruses and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.
Performance characteristics for SARS-CoV-2 were established during the 2022 SARS-CoV-2 pandemic when SARS-CoV-2 Omicron was the predominant SARS-CoV-2 variant in circulation. When other SARS-CoV-2 virus variant are emerging, performance characteristics may vary.
This test is intended for prescription use only and can be used in Point-of-Care settings. | management decisions. Positive results do not rule out co-infection with other respiratory pathogens.
Performance characteristics for SARS-CoV-2 were established during the 2021-2022 SARS-CoV-2 pandemic when SARS-CoV-2 Omicron was the predominant SARS-CoV-2 variant in circulation. When other SARS-CoV-2 virus variant are emerging, performance characteristics may vary.
This test is intended for prescription use only and can be used in Point-of-Care settings. |
| Regulation Number | 21 CFR 866.3982 | Same |
| Indication Type | In vitro diagnostic | Same |
| Intended Use Population | Individuals with symptoms of COVID-19 | Same |
| Intended Use Setting | Point-of-Care | Same |
| Specimen Type | Direct anterior nasal swab | Same |
| Analyte | SARS-CoV-2 nucleocapsid protein | Same |
| Test Type | Lateral flow immunochromatographic | Same |
| Test Result Type | Qualitative | Same |
| Test Time | 15 minutes | Same |
| General Device Characteristic Differences | | |
| Instrumentation | None | Sofia 2 Instrument |
| Result Interpretation | Visually read - visual interpretation of the presence or absence of colored line(s) on the control and test line(s) of the test strip is used to determine Positive, Negative, or Invalid results. | Instrument read - the Sofia 2 instrument scans the test strip and measures the fluorescent signal by processing the results using method specific algorithms. Sofia 2 displays the test results (Positive, Negative, or Invalid) on the screen. |
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VI Standards/Guidance Documents Referenced:
| Document Number | Title | Publishing Organization | Applicable Study |
| --- | --- | --- | --- |
| 21 CFR 866.3982 | Simple Point-Of-Care Device To Directly Detect Sars-Cov-2 Viral Targets From Clinical Specimens In Near-Patient | FDA | All |
| OMB:0910-0595 | U.S. FDA's SARS-CoV-2 Emergency Use Authorization Antigen Template for Test Developers (Dated 06 Oct 2021) | FDA | All |
| EP12-A2 | User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline-Second Edition (Sections 7.1 Controls, Section 8 Bias and Imprecision Studies (with focus on 8.3), and Appendix: Statistical Reasoning for Precision Experiment Conclusions) | CLSI | Precision/Repeatability and Reproducibility |
| EP-37 | Supplemental Tables for Interference Testing in Clinical Chemistry | CLSI | Interference |
| BS EN ISO 23640:2015 | In vitro diagnostic medical devices. Evaluation of stability of in vitro diagnostic reagents | BSI Standard | Shelf-life |
| N/A | Guidance for Industry and FDA Staff, Establishing the Performance Characteristics of In Vitro Diagnostic Devices for the Detection or Detection and Differentiation of Influenza Viruses (15 Jul 2011; Section 9.A.i) | FDA | Limit of Detection |
| N/A | Recommendations for Clinical Laboratory Improvement Amendments of 1988 (CLIA) Waiver Applications for Manufacturers of In Vitro Diagnostic Devices; Guidance for Industry and Food and Drug Administration Staff (dated 26 Feb 2020; Section IV.A. Tier I: Risk Analysis and Flex Studies) | FDA | Cross-reactivity and Microbial Interference; Development Read Time and Test Result Stability |
| N/A | Testing for Biotin Interference in In Vitro Diagnostic Devices | FDA | Biotin Interference |
VII Performance Characteristics (if/when applicable):
A Analytical Performance:
1. Precision/Reproducibility:
a) Precision and Repeatability
In the precision/repeatability (P/R) study, two blinded operators evaluated negative and contrived positive samples prepared with Gamma-irradiated SARS-CoV-2 in negative clinical matrix (NCM) at four (4) concentrations: true negative ("TN", composed of NCM only), high negative ("HN", 0.1x LoD), low positive ("LP", 1x LoD), and positive ("P", 4x LoD) (Table 1). Each operator tested two sample replicates per concentration twice daily for 12 days, resulting in a total of 96 replicates per sample level (2 operators x
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2 sample replicates x 2 sample runs daily x 12 days). Precision testing was conducted in accordance with the Package Insert. One lot of test devices was used.
Table 1. Precision and Repeatability Study - Test Sample Panel
| Spiking Concentrations | | | |
| --- | --- | --- | --- |
| Level | Concentration (x LoD) | Concentration (TCID50/mL) | Concentration on Swab (TCID50) |
| Negative Sample (TN) | NCM Only | N/A | N/A |
| High Negative (HN) | 0.1 x LoD | 7.0 x 101 | 3.5 |
| Low Positive (LP) | 1 x LoD | 7.0 x 102 | 35 |
| Positive (P) | 4 x LoD | 2.8 x 103 | 140 |
Data were analyzed by calculating percent agreement between the test results and the expected results for the sample. Results are summarized below and in Table 2:
- True negative samples produced $100\%$ expected negative agreement (96/96)
High negative samples produced $96.9\%$ expected positive agreement (93/96)
- Low positive samples produced $95.8\%$ expected positive agreement (92/96)
- Positive samples produced $100\%$ expected positive agreement (96/96)
- Out of 384 replicates tested, there were zero (0) invalid results
Table 2. Precision and Repeatability Study - Results
| Level | P (4x LoD) | | LP (1x LoD) | | HN (0.1x LoD) | | TN (NCM only) | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Operator | 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 |
| Run 1 | 24/24 | 24/24 | 24/24 | 23/24 | 24/24 | 23/24 | 24/24 | 24/24 |
| Run 2 | 24/24 | 24/24 | 22/24 | 23/24 | 23/24 | 23/24 | 24/24 | 24/24 |
| Total | 48/48 | 48/48 | 46/48 | 46/48 | 47/48 | 46/48 | 48/48 | 48/48 |
| % Agreement | 100% | 100% | 95.8 | 95.8 | 97.9 | 95.8 | 100% | 100% |
| 95% CI | 92.6-100% | 92.6-100% | 86.0-98.9% | 86.0-98.9% | 89.1-99.6% | 86.0-98.9% | 92.6-100% | 92.6-100% |
| Total | 96/96 | | 92/96 | | 93/96 | | 96/96 | |
| Total % Agreement | 100% | | 95.8% | | 96.9% | | 100% | |
| Total 95% CI | 92.6 - 100% | | 89.8 - 98.4% | | 91.2 - 98.9% | | 92.6 - 100% | |
# b) Reproducibility
The reproducibility study was designed to evaluate lot-to-lot, operator-to-operator, site-to-site, and analyte level-to-level variability to demonstrate that the Nano-Check COVID-19 Antigen Test can be performed consistently and correctly. The study was conducted at three (3) external CLIA-waived point-of-care (PoC) sites with a total of seven (7) untrained operators and at one (1) internal site with a total of three (3) operators. A total of six (6) lots of test devices were used. Blinded operators tested a coded panel of contrived samples at four analyte levels (refer to Table 1 above), with three (3) replicates per level per operator being tested across five (5) days. Per analyte level, 150 replicates were tested (10 operators x 5 days of testing x 3 replicates per day) for a total of 600 results. The results, stratified by site, are summarized below and in Table 2. No significant differences between sites, or between trained and untrained operators were observed.
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- Out of 600 samples tested, there were zero (0) invalid test results obtained
- The true negative samples produced an overall 100.0% expected negative agreement (150/150)
- The high negative (0.1x LoD) samples produced an overall 100% expected negative agreement (150/150)
- The low positive (1x LoD) samples produced an overall 98.0% expected positive agreement (147/150)
- The moderate positive (4x LoD) samples produced an overall 100% expected positive agreement (150/150)
Table 3. Reproducibility Study Results
| Site | Operator | True Negative | High Negative (0.1x LoD) | Low Positive (1x LoD) | Positive (4x LoD) |
| --- | --- | --- | --- | --- | --- |
| Site 1 (External) | n=3 Untrained | 45/45 | 45/45 | 42/45 | 45/45 |
| Site 2 (External) | n=2 Untrained | 30/30 | 30/30 | 30/30 | 30/30 |
| Site 3 (External) | n=2 Untrained | 45/45 | 45/45 | 30/30 | 45/45 |
| Site 4 (Internal) | n=3 Trained | 45/45 | 45/45 | 45/45 | 45/45 |
| Total | | 150/150 | 150/150 | 147/150 | 150/150 |
| % Agreement | | 100% | 100% | 98.0% | 100% |
| 95% CI | | 97.5-100% | 97.5-100% | 94.3-99.3% | 97.5-100% |
2. Linearity:
This study is not applicable as this test device is a qualitative assay.
3. Analytical Specificity/Interference:
a) Cross-Reactivity and Microbial Interference Study
Cross-reactivity and potential microbial interference were evaluated by testing various bacteria (10), viruses (31), fungus (1), and negative matrix (1) with the Nano-Check COVID-19 Antigen Test. Each organism and virus was tested in three (3) replicates in the presence or absence of SARS-CoV-2 (heat-inactivated, isolate hCoV-19/USA/MDHP20874/2021, Omicron variant, lineage B.1.1.529). For test kit format A (pre-filled reagent tubes), positive samples were tested at 2x LoD. For test kit format B (buffer ampules and empty reagent tubes), positive samples were tested at 3x LoD. Three blinded operators tested the samples using one lot of each test device. Cross-reactivity and interference were evaluated by the agreement with the expected negative or positive results, respectively. None of the evaluated organisms and viruses demonstrated cross-reactivity or interference with the assay at the tested concentrations. Results are summarized in Table 4 below.
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Table 4. Cross-Reactivity and Microbial Interference Results
| Microorganism / Virus | Strain | Conc. | Cross-Reactivity (Neg. agreement: SCV-2 negative reps* / all reps) | | Interference (Pos. agreement: SCV-2 positive reps / all reps) | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Kit Format A | Kit Format B | Kit Format A | Kit Format B |
| Adenovirus | Type 2 (Species C) | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Adenovirus | Type 1 (Adenoid 71) | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Mastadenovirus B | Gomen | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Coronavirus | 229E | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Coronavirus | OC43 | 4.45x104TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Coronavirus | NL63 | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| MERS-CoV | EMC/2012 | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| SARS-CoV | Urbani | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Metapneumovirus | TN/83-1211 | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Parainfluenza Virus Type 1 | HPIVI/FRA/29221106/2009 | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Parainfluenza Virus Type 2 | Greer | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Parainfluenza Virus Type 3 | NIH 47885 | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Parainfluenza Virus Type 4B | 19503 | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Influenza A H1N1 | New Caledonia/20/1999 | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Influenza A H1N1 | San Diego/1/2009 | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Influenza A H3N2 | Victoria/361/2011 | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Influenza A H3N2 | Wisconsin/67/2005 | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Influenza B Virus | Brisbane/60/2008 (Victoria) | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Influenza B Virus | Texas/06/2011 (Yamagata) | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Influenza B Virus | B/GL/1739/54 | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Enterovirus 71 | MP4 | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Enterovirus Species D | Type 68, USA/2018-23087 | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Respiratory Syncytial Virus | Strain long | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Respiratory Syncytial Virus | Type A (2001/2-20) | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Respiratory Syncytial Virus | Type A (1998/12-21) | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
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| Microorganism / Virus | Strain | Conc. | Cross-Reactivity (Neg. agreement: SCV-2 negative reps* / all reps) | | Interference (Pos. agreement: SCV-2 positive reps / all reps) | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Kit Format A | Kit Format B | Kit Format A | Kit Format B |
| Respiratory Syncytial Virus | Type B1 | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Rhinovirus 20 | 15-CV19 | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Measles Virus | Edmonston | 1.7x104TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Mumps Virus | MuV/Iowa.US/2006 | 1.0x105TCID50/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Coxsackie Virus | B4 | 1.0x105U/mL TCID50 | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Epstein-Barr Virus | B95-B | 1.0x105cp/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Haeomophilus influenzae | Type B (Eagan) | 1.0x106CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Streptococcus pneumoniae, Type 19F | Strain Z022 | 1.0x106CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Streptococcus pyogenes | Bruno | 1.0x106CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Streptococcus salivarius | N/A | 1.0x106CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Candida albicans | Z006 | 1.0x106CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Bordetella pertussis | Strain 5 | 1.0x106CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Mycoplasma pneumoniae | N/A | 1.0x106CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Chlamydia pneumoniae | TW-183 | 1.0x106CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Legionella pneumophila | N/A | 1.0x106CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Staphylococcus aureus | MRSA, COL | 1.0x106CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Staphylococcus epidermidis | MRSE, RP62A | 1.0x106CFU/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Negative clinical matrix | N/A | N/A | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
* Reps = Replicates
Kit Format A includes pre-filled reagent tubes
Kit Format B includes buffer-filled ampules and empty reagent tubes
The sponsor additionally tested coronavirus HKU1 (HCoV-HKU1) using 20 positive clinical specimens. Clinical samples were diluted with negative clinical matrix and tested in the same manner as the viruses above. No cross-reactivity or interference was observed for any of the tested samples.
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# b) Endogenous and Exogenous Interfering Substances Study
This study evaluated endogenous or exogenous substances that may potentially interfere with the performance of the Nano-Check COVID-19 Antigen Test. Each potentially interfering substance was tested in triplicate in negative clinical matrix at targeted concentrations in the presence or absence of 3x LoD SARS-CoV-2 (heat-inactivated, isolate hCoV-19/USA/MD-HP20874/2021, Omicron variant, lineage B.1.1.529). Three blinded operators tested the samples using one lot of kit format A (pre-filled reagent tubes) and one lot of kit format B (buffer ampules and empty reagent tubes). The effects of the substances were evaluated by the agreement with the expected positive or negative results. None of the evaluated substances demonstrated interference with the assay at the tested concentrations. Results are summarized in Table 5 below.
Table 5. Interfering Substances Results
| Substance | Active Ingredient of Substance | Interferent Conc. | Negative Agreement (SCV-2 negative reps* / all reps) | | Positive Agreement (SCV-2 positive reps / all reps) | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Kit Format A | Kit Format B | Kit Format A | Kit Format B |
| Nasal Spray – Afrin | Oxymetazoline | 15% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Nasal Spray – NasalCrom | Cromolyn sodium | 15% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Nasal Spray – Flonase | Fluticasone propionate | 15% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Nasal Spray – CVS | Phenylephrine HCl | 15% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Budesonide Nasal Spray | Budenoside | 15% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Oral Pain Reliever Spray (Chloraseptic) | Phenol, Menthol | 15% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Orrivine Nasal Drops | Xylometazoline | 15% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Walgreens Zinc Lozenges | Zincum gluconicum | 5% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Similasan Nasal Allergy Relief | Cardiospermun, Galphimia glauca, Luffa operculate, Sabadilla | 15% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| NeilMed NasoGEL | Sodium hyaluronate, Allantoin, NaCl, Methylparaben, Propylparaben | 15% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Cepacol Sore Throat Lozenges | Benzocaine, Menthol | 15% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Alkalol Homeopathic Allergy Nasal Spray | N/A | 15% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| NASONEX 24 hr Allergy | Mometasone furoate monohydrate | 15% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Nasacort Allergy 24 hr | Triamcinolone acetonide | 15% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
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| Substance | Active Ingredient of Substance | Interferent Conc. | Negative Agreement (SCV-2 negative reps* / all reps) | | Positive Agreement (SCV-2 positive reps / all reps) | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Kit Format A | Kit Format B | Kit Format A | Kit Format B |
| Histaminum 30C | Histaminum hydrochloricum 30C HPUS | 5% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Zicam Oral Mist | Zincum aceticum, Zincum gluconicum | 15% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Tobramycin | Tobramycin | 33 μg/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Mupirocin | Mupirocin | 1.5 μg/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Oseltamivir Phosphate | Oseltamivir Phosphate | 0.39 μg/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Beclomethasone dipropionate | Beclomethasone dipropionate | 5.04 μg/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Dexamethasone | Dexamethasone | 12 μg/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Flunisolide | Flunisolide | 87 μg/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Remdesivir | Remdesivir | 240 μg/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Molnupiravir | Molnupiravir | 32.9 μg/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Softsoap Hand Soap | N/A | 10% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| CVS Hand Sanitizer Gel | 70% Ethyl Alcohol | 1% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Bovine Mucin | Mucin protein | 2.5 mg/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Purified Human Neutrophils | N/A | 5x106cells/mL | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
| Human Whole Blood | N/A | 2.5% (v/v) | 100% (3/3) | 100% (3/3) | 100% (3/3) | 100% (3/3) |
* Reps = Replicates
Kit Format A includes pre-filled reagent tubes
Kit Format B includes buffer-filled ampules and empty reagent tubes
c) Biotin Interference Study
The Nano-Check COVID-19 Antigen Test is based on a sandwich assay format that utilizes a biotin-streptavidin complex. The sponsor conducted a biotin interference study to assess if free biotin (vitamin B7) in samples may compete with the biotinylated complex and impact assay results. Biotin was tested at five concentrations in triplicate in negative clinical matrix in the presence of 3x LoD SARS-CoV-2 (heat-inactivated, isolate hCoV-19/USA/MD-HP20874/2021, Omicron variant, lineage B.1.1.529). One lot of kit format A (pre-filled reagent tubes) and one lot of kit format B (buffer ampules and empty reagent tubes) was used for testing. The effects of biotin interference were evaluated by the agreement with the expected positive results. Interference was not observed at any of the concentrations tested. Results are summarized in Table 6 below.
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Table 6 Biotin Interference Results
| Concentration of Biotin (ng/mL) | Concentration of SARS-CoV-2 | Positive Agreement (SCV-2 positive reps */ all reps) | |
| --- | --- | --- | --- |
| | | Kit Format A | Kit Format B |
| 3500 | 3x LoD | 100% (5/5) | 100% (5/5) |
| 2400 | 3x LoD | 100% (5/5) | 100% (5/5) |
| 1200 | 3x LoD | 100% (5/5) | 100% (5/5) |
| 600 | 3x LoD | 100% (5/5) | 100% (5/5) |
| 300 | 3x LoD | 100% (5/5) | 100% (5/5) |
| 150 | 3x LoD | 100% (5/5) | 100% (5/5) |
* Reps = Replicates
Kit Format A includes pre-filled reagent tubes
Kit Format B includes buffer-filled ampules and empty reagent tube
4. Assay Reportable Range:
This section is not applicable as this test device is a qualitative assay.
5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):
a) Internal Controls
The Nano-Check COVID-19 Antigen Test has a built-in internal procedural control. When sample is added, colloidal gold-conjugated chicken antibodies in the dye pad will mix with the sample and migrate through the test strip. The labeled antibody complexes will bind with the goat anti-chicken antibodies immobilized on the control line on the nitrocellulose membrane. This will form a colored control line in the control line region. Formation of the control line serves as an internal control indicating proper sample application procedure and membrane wicking has occurred, and indicates reagents are functioning appropriately.
b) External Controls
The Nano-Check COVID-19 Antigen Test includes two external controls. The external positive control swab contains recombinant SARS-CoV-2 nucleocapsid protein antigen equivalent to 2x LoD on the swab head. The external positive control swab is intended to mimic positive samples to ensure the test device is functioning as expected. The external negative control swab contains blank Universal Transport media and is intended to mimic negative samples to ensure the test device is functioning as expected.
Lot-to-lot reproducibility of the external positive and negative control swabs was evaluated with three lots of each external control and three blinded operators. Ten replicates from each lot were tested on a unique lot of test kit. For each external control lot, all positive and negative controls produced 100% agreement with the expected results, as summarized in the table below.
Table 7. Validation of External Control Materials
| External Control | Lot No. | # Neg. | # Pos. | % Expected Agreement |
| --- | --- | --- | --- | --- |
| | 0804-CN-2B01-P | 0/10 | 10/10 | 100% |
| | 0804-CN-2D29-P | 0/10 | 10/10 | 100% |
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| External Control | Lot No. | # Neg. | # Pos. | % Expected Agreement |
| --- | --- | --- | --- | --- |
| External Positive Control | 0804-CN-2E04-P | 0/10 | 10/10 | 100% |
| External Negative Control | 0804-CN-2B01-N | 10/10 | 0/10 | 100% |
| | 0804-CN-2D29-N | 10/10 | 0/10 | 100% |
| | 0804-CN-2E04-N | 10/10 | 0/10 | 100% |
## c) Reagent Stability/Shelf-Life
(1) In order to determine the shelf-life of the Nano-Check COVID-19 Antigen Test in the intended storage conditions (2-30°C), a real-time stability study was conducted using three (3) lots of test kit stored at 2-8°C, room temperature, and 30°C after manufacturing. Single test kits (Format A) were assessed with negative samples and contrived positive samples prepared using inactivated SARS-CoV-2 omicron variant at 3x LoD in NCM. Five replicates per timepoint per storage condition were analyzed. Three lots of external control swabs were also stored in the same conditions and analyzed along with the contrived positive and negative samples. Data collected to date support a test kit and external control shelf-life of 14 months.
(2) Separately, the shelf-life of ampules containing reagent buffer (included in the multi test kit Format B) was evaluated by storing three lots of ampules at 2-8°C, room temperature, and 30°C after manufacturing and testing them with a single lot of test devices stored in the same condition. Ten (10) replicates of negative samples and contrived positive samples prepared using inactivated SARS-CoV-2 omicron variant at 2x LoD were tested per timepoint per storage condition. Data collected to date support an ampule shelf-life of 17 months.
## d) Specimen Stability
Specimen stability was assessed using contrived positive samples prepared with nasal fluid and SARS-CoV-2 (omicron variant, isolate hCoV-19/USA/MD-HP20874/2021). Four test samples were prepared: true negative (TN, negative nasal fluid with no analyte), high negative (HN, 0.3x LoD), low positive (LP, 1x LoD), and positive (P, 3x LoD). Samples were stored at ambient temperature (23.5°C), high room temperature (30°C), and refrigerated (2-8°C) and tested at 1, 2, 4, 8, 24, 48, and 72 hours. The ambient temperature condition was used for time 0 testing of all samples. Five replicates of each concentration were tested in accordance with the package insert at each timepoint for each condition. Positive percent agreement summary results are included in the table below.
| Specimen Storage Parameters | Conc. | % positive (SCV-2 positive reps* / all reps) | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | 0 hrs. | 1 hr. | 2 hrs. | 4 hrs. | 8 hrs. | 24 hrs. | 48 hrs. | 72 hrs. |
| Ambient Temp. (23.5°C) | TN | 0.0% (0/0) | 0.0% (0/0) | 0.0% (0/0) | 0.0% (0/0) | 0.0% (0/0) | 0.0% (0/0) | 0.0% (0/0) | 0.0% (0/0) |
| | HN | 40.0% (2/5) | 40.0% (2/5) | 40.0% (2/5) | 40.0% (2/5) | 40.0% (2/5) | 20.0% (1/5) | 0.0% (0/0) | 0.0% (0/0) |
| | LP | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) | 60.0% (3/5) | 20.0% (1/5) |
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| Specimen Storage Parameters | Conc. | % positive (SCV-2 positive reps* / all reps) | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | 0 hrs. | 1 hr. | 2 hrs. | 4 hrs. | 8 hrs. | 24 hrs. | 48 hrs. | 72 hrs. |
| | P | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) |
| High Room Temp. (30°C) | TN | N/A | 0.0% (0/0) | 0.0% (0/0) | 0.0% (0/0) | 0.0% (0/0) | 0.0% (0/0) | 0.0% (0/0) | 0.0% (0/0) |
| | HN | N/A | 40.0% (2/5) | 40.0% (2/5) | 20.0% (1/5) | 20.0% (1/5) | 0.0% (0/0) | 0.0% (0/0) | 0.0% (0/0) |
| | LP | N/A | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) | 20.0% (1/5) | 0.0% (0/0) |
| | P | N/A | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) |
| Refrigerated (2-8°C) | TN | N/A | 0.0% (0/0) | 0.0% (0/0) | 0.0% (0/0) | 0.0% (0/0) | 0.0% (0/0) | 0.0% (0/0) | 0.0% (0/0) |
| | HN | N/A | 60.0% (3/5) | 40.0% (2/5) | 60.0% (3/5) | 60.0% (3/5) | 40.0% (2/5) | 40.0% (2/5) | 0.0% (0/0) |
| | LP | N/A | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) |
| | P | N/A | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) | 100% (5/5) |
* Reps = Replicates
Based on the study results, the following storage conditions of nasal swabs are supported:
- 8 hours when stored at ambient room temperature (23.5°C)
- 8 hours when stored at high room temperature (30°C)
- 48 hours when stored refrigerated (2-8°C)
6. Detection Limit:
Kit Format A - LoD
The Limit of Detection (LoD) of the Nano-Check COVID-19 Antigen Test kit format A (multi test packaging with pre-filled reagent tube) was determined by evaluating different dilutions of inactivated SARS-CoV-2 stocks in negative clinical matrix. The following SARS-CoV-2 strains were used: Gamma-irradiated SARS-CoV-2 isolate USA-WA1/2020, heat-inactivated SARS-CoV-2 isolate USA/MD-HP20874/2021 (Omicron variant, lineage B.1.1.529) and UV-inactivated SARS-CoV-2 isolate USA/PHC658/2021 (Delta variant, lineage B.1.6172.2). Pooled human nasal wash was used as the negative clinical matrix.
For range finding preliminary LoD determination of SARS-CoV-2 USA-WA1/2020, five (5) 10-fold serial dilutions were made from SARS-CoV-2 USA-WA1/2020 stock into negative clinical matrix. Three replicates were tested for each of the five dilutions to determine the preliminary LoD concentration on one lot of the Nano-Check COVID-19 Antigen Test. The preliminary LoD was further refined using three (3) 2-fold serial dilutions with three replicates each. Thereafter, the LoD was confirmed by testing 20 replicates at the preliminary LoD concentration. LoD confirmatory testing was repeated with a second lot of Nano-Check COVID-19 Antigen Test.
For range finding preliminary LoD determination of the other SARS-CoV-2 isolates, three 10-fold serial dilutions and then 2-fold serial dilutions of SARS-CoV-2 were used to determine the preliminary LoD. The LoD was confirmed by testing 20 replicates at the
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preliminary LoD concentration. A single lot of Nano-Check COVID-19 Antigen Test was used for LoD testing with the additional virus strains.
Data are summarized in Table 9. The Nano-Check COVID-19 Antigen Test LoD for the following SARS-CoV-2 strains was confirmed to be:
USA-WA1/2020: $7.0 \times 10^{2} \mathrm{TCID}_{50} / \mathrm{mL}$
USA/MD-HP20874/2021 (Omicron variant, linage B.1.1.529): $1.95 \times 10^{2} \mathrm{TCID}_{50} / \mathrm{mL}$
USA/PHC658/2021 (Delta variant, lineage B.1.6172.2): $5.21 \times 10^{2} \mathrm{TCID}_{50} / \mathrm{mL}$
Table 9. Limit of Detection Study Results (Kit Format A)
| Isolate | Lot | Conc. (TCID50/mL) | Conc. (TCID50/swab) | Range Finding | | | Confirmation | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | n | Neg | Pos (% Pos) | n | Neg | Pos (% Pos) |
| USA-WA1/2020 | Lot 1 | 2.8x105 | 1.4x104 | 3 | 0 | 3 (100%) | | | |
| | | 2.8x104 | 1.4x103 | 3 | 0 | 3 (100%) | | | |
| | | 2.8x103 | 1.4x102 | 3 | 0 | 3 (100%) | | | |
| | | 2.8x102 | 1.4x101 | 3 | 3 | 0 (0%) | | | |
| | | 2.8x101 | 1.4 | 3 | 3 | 0 (0%) | | | |
| | | 1.4x103 | 70 | 3 | 0 | 3 (100%) | | | |
| | | 7.0x102 | 35 | 3 | 0 | 3 (100%) | 20 | 0 | 20 (100%) |
| | 3.5x102 | 17.5 | 3 | 3 | 0 (0%) | | | | |
| | Lot 2 | 1.4x103* | 70 | 0 | - | - | 20 | 0 | 20 (100%) |
| | | 7.0x102* | 35 | 0 | - | - | 20 | 1 | 19 (95%) |
| 3.5x102* | | 17.5 | 0 | - | - | 20 | 3 | 17 (85%) | |
| USA/MD-HP20874/2021 (Omicron) | Lot 2 | 3.89x103 | 1.9x102 | 3 | 0 | 3 (100%) | | | |
| | | 3.89x102 | 1.9x101 | 3 | 0 | 3 (100%) | 20 | 0 | 20 (100%) |
| | | 3.89x101 | 1.9 | 3 | 3 | 0 (0%) | | | |
| | | 1.95x102 | 9.75 | 3 | 0 | 3 (100%) | 20 | 1 | 19 (95%) |
| | | 9.75x101 | 4.9 | 3 | 1 | 2 (66.6%) | 20 | 7 | 13 (65%) |
| USA/PHC658/2021 (Delta) | Lot 2 | 4.17x104 | 2x103 | 3 | 0 | 3 (100%) | | | |
| | | 4.17x103 | 2x102 | 3 | 0 | 3 (100%) | | | |
| | | 4.17x102 | 2x101 | 3 | 2 | 1 (33.3%) | | | |
| | | 2.09x103 | 1.04x102 | 3 | 0 | 3 (100%) | | | |
| | | 1.04x103 | 52 | 3 | 0 | 3 | 20 | 0 | 20 |
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| Isolate | Lot | Conc. (TCID50/mL) | Conc. (TCID50/swab) | Range Finding | | | Confirmation | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | n | Neg | Pos (% Pos) | n | Neg | Pos (% Pos) |
| | | | | | | (100%) | | | (100%) |
| | | 5.21x102 | 26 | 3 | 0 | 3 (100%) | 20 | 1 | 19 (95%) |
| | | 2.61x102 | 13 | 0 | - | - | 20 | 9 | 11 (55%) |
# Kit Format B LoD
The LoD of the Nano-Check COVID-19 Antigen Test kit format B (buffer ampule and empty reagent tube) was evaluated and compared to the LoD for kit format A. Dilutions of inactivated SARS-CoV-2 (isolate USA/MD-HP20874/2021, Omicron variant, linage B.1.1.529) prepared in negative clinical matrix (pooled human nasal wash) were tested with test kit formats.
An initial range finding study was conducted with kit format B to identify a tentative LoD using four 10-fold serial dilutions of SARS-CoV-2 in negative clinical matrix. Three replicates were tested for each of the five dilutions to determine the preliminary LoD concentration on one lot of the Nano-Check COVID-19 Antigen Test kit format B. The preliminary LoD was further refined using three 2-fold serial dilutions with three replicates each.
Data are summarized in Table 10 below. The Nano-Check COVID-19 Antigen Test LoD for kit format A and kit format B was confirmed to be $1.95 \times 10^{2} \mathrm{TCID}_{50} / \mathrm{mL}$ with SARS-CoV-2 USA/MD-HP20874/2021 (Omicron variant, linage B.1.1.529). The test kit formats have equivalent LoD.
Table 10. Limit of Detection Study (Kit Format B)
| Isolate | Conc. (TCID50/mL) | Conc. (TCID50/swab) | Range Finding | | | Confirmation | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | n | Neg | Pos (% Pos) | n | Neg | Pos (% Pos) |
| USA/MD-HP20874/2021 (Omicron) | 3.89 x 102 | 1.9x101 | 3 | 0 | 3 (100%) | 20 | 0 | 20 (100%) |
| | 1.95 x 102 | 9.75 | 3 | 0 | 3 (100%) | 20 | 1 | 19 (95%) |
| | 9.73 x 101 | 4.8 | 3 | 1 | 3 (100%) | 20 | 6 | 14 (70%) |
| | 4.86 x 101 | 2.4 | 3 | 2 | 0 (0%) | | | |
| | 3.89 x 101 | 1.9 | 3 | 3 | 0 (0%) | | | |
| | 3.89 x 100 | 1.9x10-1 | 3 | 3 | 3 (100%) | | | |
| | 3.89 x 10-1 | 1.9x10-2 | 3 | 3 | 3 (100%) | | | |
# WHO International Standard LoD
The LoD of the Nano-Check COVID-19 Antigen Test was also determined by evaluating different dilutions of the WHO International Standard for SARS-CoV-2 antigen (NIBSC
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code: 21/368) in negative clinical swab matrix. The WHO International Standard for SARS-CoV-2 containing lyophilized SARS-CoV-2 antigen was reconstituted in 0.25 mL of ultrapure water (for a final concentration 20,000 IU/mL). The LoD was determined as the lowest virus concentration that was detected ≥ 95% of the time (i.e., concentration at which at least 19 out of 20 replicates tested positive). The limit of detection was established in two phases.
Three-fold serial dilutions were made from the WHO International Standard for SARS-CoV-2 antigen into negative clinical matrix (nasal fluid). Three replicates were tested on one lot of the assay for each dilution to determine the preliminary LoD concentration of the device. The lowest concentration with 3/3 positive results from each lot was considered the preliminary LoD. For each replicate, 50 μL of virus dilution was applied to a swab and the swab was processed according to the IFU. The results are summarized below.
Table 11. WHO International Standard for SARS-CoV-2 Antigen Preliminary LoD Results
| Concentration of WHO International Standard for SARS-CoV-2 Antigen (Sample) | Amount of WHO International Standard for SARS-CoV-2 Antigen on Dry Swab | # Positive / # Tested |
| --- | --- | --- |
| 4,000 IU/mL | 200 IU/swab | 3/3 |
| 1,333 IU/mL | 67 IU/swab | 3/3 |
| 444 IU/mL | 22 IU/swab | 1/2 |
| 148 IU/mL | 7.4 IU/swab | 0/3 |
| 49 IU/mL | 2.5 IU/swab | 0/3 |
The preliminary LoD concentration was tested with an additional 17 replicates, for a total of 20 replicates, to confirm the LoD. Concentrations above and below the preliminary LoD were also tested with 20 replicates to further refine the LoD. Samples were prepared as for the preliminary LoD study above. To confirm the LoD, at least 19 of 20 replicates should be positive. The results are summarized below.
Table 12. WHO International Standard for SARS-CoV-2 Antigen Confirmatory LoD Results
| Concentration of WHO International Standard for SARS-CoV-2 Antigen (Sample) | Amount of WHO International Standard for SARS-CoV-2 antigen on Dry Swab | # Positive / # Tested |
| --- | --- | --- |
| 2,000 IU/mL | 100 IU/swab | 20/20 |
| 1,333 IU/mL | 67 IU/swab | 19/20 |
| 1,000 IU/mL | 50 IU/swab | 20/20 |
| 889 IU/mL | 44 IU/swab | 19/20 |
| 800 IU/mL | 40 IU/swab | 16/20 |
The LoD for the Nano-Check COVID-19 Antigen Test using the 1st WHO International Standard for SARS-CoV-2 antigen (NIBSC code: 21/368) in nasal matrix was determined to be 889 IU/mL (44 IU/swab).
7. High-dose Hook Effect Study
A high-dose hook effect study was conducted to determine if a hook effect would be observed at high analyte concentration (i.e., a false negative at high concentrations of SARS-CoV-2). Five concentrations of SARS-CoV-2 (isolate USA-WA1/2020) were tested with three replicates each using a single lot of test kit format A (pre-filled reagent tube).
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Concentrations ranged from 4,000x LoD (the maximum virus concentration possible, undiluted from the viral stock) to 1x LoD (7.0x10² TCID₅₀/mL) diluted in negative clinical matrix. All tested samples demonstrated 100% positivity, as expected (Table 13). The Nano-Check COVID-19 Antigen Test did not display a Hook Effect for the tested SARS-CoV-2 concentrations.
Table 13. High-Dose Hook Effect Study Results
| Conc. (TCID_{50}/mL) | Sample Level | Positive Agreement (SCV-2 positive reps* / all reps) |
| --- | --- | --- |
| 2.8 x 10^{6} | 4000x LoD | 100% (3/3) |
| 2.8 x 10^{5} | 400x LoD | 100% (3/3) |
| 2.8 x 10^{4} | 40x LoD | 100% (3/3) |
| 2.8 x 10^{3} | 4x LoD | 100% (3/3) |
| 7.0 x 10^{2} | 1x LoD | 100% (3/3) |
* Reps = replicates
## 8. Inclusivity (Analytical Reactivity)
An inclusivity study was performed to demonstrate that the Nano-Check COVID-19 Antigen Test can detect circulating SARS-CoV-2 strains/isolates. Fifteen (15) stock strains and/or clinical isolates were included. A preliminary study was conducted to determine a near cutoff concentration for each strain/isolate tested in triplicate with one lot of test kit format A. Three-fold dilution series using the preliminary LoD were then prepared, and five replicates were tested to identify the dilution at which assay reactivity decreased for each strain/isolate using a separate lot of test kit format A (results summarized in Table 14). All tested isolates were detected.
Table 14. Inclusivity Study Results
| Variant / Lineage | Strain | Concentration | Positive Agreement (SCV-2 pos. reps / all reps) |
| --- | --- | --- | --- |
| Wild-type | USA-WA1/2020* | 4.79 x 10^{4} TCID_{50}/mL | 100% (5/5) |
| | | 1.60 x 10^{4} TCID_{50}/mL | 80.0% (4/5) |
| | | 5.32 x 10^{3} TCID_{50}/mL | 0% (0/5) |
| Alpha (B.1.1.7) | USA/CA_CDC_5574/2020 | 2.03 x 10^{7} genome copies/mL | 100% (5/5) |
| | | 6.77 x 10^{6} genome copies/mL | 100% (5/5) |
| | | 2.26 x 10^{6} genome copies/mL | 40.0% (2/5) |
| | England/204820464/2020 | 7.19 x 10^{3} TCID_{50}/mL | 100% (5/5) |
| | | 2.40 x 10^{3} TCID_{50}/mL | 20.0% (1/5) |
| | | 7.99 x 10^{2} TCID_{50}/mL | 0% (0/5) |
| | USA/CA_CDC_5574/2020 | 2.39 x 10^{4} TCID_{50}/mL | 100% (5/5) |
| | | 7.97 x 10^{3} TCID_{50}/mL | 80.0% (4/5) |
| | | 2.66 x 10^{3} TCID_{50}/mL | 20.0% (1/5) |
| Beta (B.1.351) | hCoV-19/USA/MD-HP01542/2021 | 2.16 x 10^{5} genome copies/mL | 100% (5/5) |
| | | 7.20 x 10^{4} genome copies/mL | 100% (5/5) |
| | | 2.40 x 10^{4} genome copies/mL | 0% (0/5) |
| | USA/MD-HP01542/2021 | 3.80 x 10^{6} genome copies/mL | 100% (5/5) |
| | | 1.27 x 10^{6} genome copies/mL | 80.0% (4/5) |
| | | 4.22 x 10^{5} genome copies/mL | 20.0% (1/5) |
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| Variant / Lineage | Strain | Concentration | Positive Agreement (SCV-2 pos. reps / all reps) |
| --- | --- | --- | --- |
| | South Africa/KRISP-K005325/2020 | 1.90 x 10^{4} TCID_{50}/mL | 100% (5/5) |
| | | 6.33 x 10^{3} TCID_{50}/mL | 60.0% (3/5) |
| | | 2.11 x 10^{3} TCID_{50}/mL | 0% (0/5) |
| Gamma (P.1) | Japan/TY7-503/2021 | 1.58 x 10^{4} TCID_{50}/mL | 100% (5/5) |
| | | 5.27 x 10^{3} TCID_{50}/mL | 40.0% (2/5) |
| | | 1.76 x 10^{3} TCID_{50}/mL | 0% (0/5) |
| | USA/NY-Wadsworth-21033899-01/2021 | 7.85 x 10^{3} TCID_{50}/mL | 100% (5/5) |
| | | 2.62 x 10^{3} TCID_{50}/mL | 60.0% (3/5) |
| | | 8.72 x 10^{2} TCID_{50}/mL | 20.0% (1/5) |
| Delta (B.1.617.2) | hCoV-19/USA/MD-HP05285/2021 | 2.16 x 10^{8} genome copies/mL | 100% (5/5) |
| | | 7.20 x 10^{7} genome copies/mL | 100% (5/5) |
| | | 2.40 x 10^{7} genome copies/mL | 20.0% (1/5) |
| | USA/PHC658/2010 | 5.21 x 10^{2} TCID_{50}/mL | 100% (5/5) |
| | | 1.74 x 10^{2} TCID_{50}/mL | 40.0% (2/5) |
| | | 5.79 x 10^{1} TCID_{50}/mL | 0% (0/5) |
| | USA/MD-HP05285/2021 | 1.50 x 10^{4} genome copies/mL | 100% (5/5) |
| | | 5.00 x 10^{4} genome copies/mL | 100% (5/5) |
| | | 1.67 x 10^{3} genome copies/mL | 20.0% (1/5) |
| Kappa (B.1.617.1) | USA/CA-Stanford-15_S02/2021 | 8.48 x 10^{4} TCID_{50}/mL | 100% (5/5) |
| | | 2.83 x 10^{4} TCID_{50}/mL | 40.0% (2/5) |
| | | 9.42 x 10^{3} TCID_{50}/mL | 0% (0/5) |
| Omicron (B.1.1529) | hCoV-19/USA/MD-HP20874/2021 | 1.95 x 10^{2} TCID_{50}/mL | 100% (5/5) |
| | | 6.50 x 10^{1} TCID_{50}/mL | 60.0% (3/5) |
| | | 2.17 x 10^{1} TCID_{50}/mL | 20.0% (1/5) |
| | USA/GA-EHC-2811C/2021 | 6.33 x 10^{4} genome copies/mL | 100% (5/5) |
| | | 2.11 x 10^{4} genome copies/mL | 100% (5/5) |
| | | 7.03 x 10^{3} genome copies/mL | 60.0% (3/5) |
*The USA-WA1/2020 strain tested here is from a different lot/supplier than the strain used for LoD testing. The stock/lot used for LoD testing is the same as that used for the remaining analytical studies. Therefore, the LoD concentration differences observed here are deemed to be a result of propagating the virus stocks and its functional testing method.
9. Assay Cut-Off:
Not applicable.
B Comparison Studies:
1. Method Comparison with Predicate Device:
Refer to Section VII.C (Clinical Studies) below for clinical validation.
2. Matrix Comparison:
The Nano-Check COVID-19 Antigen Test is only intended for qualitative detection of nucleocapsid protein antigen from SARS-CoV-2 in direct anterior nasal swab specimens. As
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no other specimen or sample type is claimed for this device, a matrix comparison study to support other sample types for clinical testing with this device was not performed.
However, the sponsor conducted a matrix equivalency study between negative clinical nasal swab matrix (nasal fluid) and representative negative clinical matrix (NCM derived from nasal wash) that was used in some analytical studies (reproducibility/precision, cross-reactivity, interference, inclusivity, and reagent/kit storage). The data demonstrated the equivalent performance of the test with both matrices.
# C Clinical Studies:
The performance of the Nano-Check COVID-19 Antigen Test in detecting SARS-CoV-2 viral nucleoprotein antigen from anterior nasal swab samples was evaluated in a multi-center, prospective study conducted in the U.S. from January 2022 to January 2023 when Omicron was the predominantly circulating variant. A total of seven hundred and eighty-six (786) subjects were consecutively enrolled and tested across four (4) different point-of-care (POC) CLIAwaived sites. Nine (9) operators across the study sites conducted the enrollment and testing of the subjects. The study only enrolled subjects with symptoms of respiratory infection consistent with SARS-CoV-2 infection.
Two anterior nasal (AN) swabs were collected from each study subject during same visit. The first swab (for comparator testing) was collected by the study operator from both sides of the nose, placed into a transport tube containing $1\mathrm{mL}$ of Universal Viral Transport (UVT) media, stored on dry ice, and shipped with dry ice to the reference laboratory. Upon receipt by the reference laboratory, the swab was tested with a highly sensitive FDA cleared RT-PCR comparator assay. The second swab was collected by the study operator from both sides of the nose and tested immediately with the Nano-Check COVID-19 Antigen Test at the site. Demographics, symptom information, and health history were also collected from each subject.
There were 670 evaluable subjects with $40.4\%$ (271/670) male and $59.6\%$ (399/670) female, with a mean age of 37 years. A total of one hundred and sixteen (116) subjects of the 786 enrolled were excluded because they did not meet the enrollment criteria. Results obtained with the Nano-Check COVID-19 Antigen Test were compared to the results obtained with the RT-PCR comparator test to determine clinical sensitivity and specificity. The study cohort included $18.37\%$ low positive samples.
The Nano-Check COVID-19 Antigen Test demonstrated a clinical sensitivity as estimated by positive precent agreement of $83.67\%$ (123/147; $95\%$ CI: $76.86\% - 88.78\%$ ) and specificity as estimated by negative percent agreement of $99.62\%$ (521/523; $95\%$ CI: $98.62\% - 99.90\%$ ) compared to the comparator method.
Table 15. Clinical performance of the Nano-Check COVID-19 Antigen Test compared to a highly sensitive cleared RT-PCR comparator
| | Cleared RT-PCR Comparator | | Total | |
| --- | --- | --- | --- | --- |
| | | Pos | | Neg |
| Nano-Check COVID-19 Antigen Test | Pos | 123 | 2 | 125 |
| | Neg | 24 | 521 | 545 |
| Total | | 147 | 523 | 670 |
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Table 16. Clinical performance of the Nano-Check COVID-19 Antigen Test stratified by DPSO
| Days Post Symptom Onset | PPA | NPA |
| --- | --- | --- |
| 1 | 80.95% (17/21) | 100.00% (74/74) |
| 2 | 80.45% (37/46) | 99.51% (202/203) |
| 3 | 84.09% (37/44) | 99.32% (145/146) |
| 4 | 94.12% (32/34) | 100.00% (96/96) |
| Total | 83.67% (123/147)
95% CI: 76.86% - 88.78% | 99.62% (521/523)
95% CI: 98.62% - 99.90% |
1. Clinical Sensitivity:
Refer to Section VII.C (Clinical Studies) above for the clinical validation, including test sensitivity/positive percent agreement (PPA). The PPA for the test is 83.67% (123/147; 95% CI: 73.86% - 88.78%).
2. Clinical Specificity:
Refer to Section VII.C (Clinical Studies) above for the clinical validation, including test specificity/negative percent agreement (NPA). The NPA for the test is 99.62% (521/523; 95% CI: 98.62% - 99.90%).
3. Serial Testing:
As a mitigation for the low performance of the device at Day 0 of symptom onset, as indicated by the clinical study data and in other studies for test devices of a similar principle and design, the Intended Use for this test device (and associated Instructions for Use) include recommendations for repeat testing (i.e., test at least twice over three days with at least 48 hours between tests). This mitigation is supported by data generated by the National Institutes for Health (NIH) and the University of Massachusetts Chan Medical School (in collaboration with the FDA) demonstrating that repeat testing over multiple days improves test performance and increases the likelihood that a COVID-19 antigen test will accurately detect an infection. These results have informed the FDA's general understanding that repeat testing after a negative result from a COVID-19 antigen test reduces the risk of a false negative result. Please refer to the following studies for additional details:
- Finding a Needle in the Haystack: Design and Implementation of a Digital Site-less Clinical Study of Serial Rapid Antigen Testing to Identify Asymptomatic SARS-CoV-2 Infection - https://www.medrxiv.org/content/10.1101/2022.08.04.22278274v1
- Performance of Screening for SARS-CoV-2 using Rapid Antigen Tests to Detect Incidence of Symptomatic and Asymptomatic SARS-CoV-2 Infection: findings from the Test Us at Home prospective cohort study - https://www.medrxiv.org/content/10.1101/2022.08.05.22278466v1
D Clinical Cut-Off:
The Clinical Cut-off study is not applicable, as there is not clinical cutoff related to the presence of SARS-CoV-2 in patient samples.
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E Expected Values/Reference Range:
Patient samples are expected to be negative for SARS-CoV-2.
F Other Supportive Performance Characteristics Data:
1. Flex Studies
To assess the robustness of the Nano-Check COVID-19 Antigen Test, flex studies were conducted that assessed all major aspects of the test procedure (sample volume, reading time, extraction buffer volume, bubbles in the reagent tube, swab elution time and procedure, sample hold time before and during processing, use of incorrect specimen type) and variability of environmental test conditions that the test may be subjected to when in use (lighting, disturbance during use, temperature and humidity stress conditions). Testing was performed with contrived positive nasal swabs generated by diluting heat inactivated SARS-CoV-2 virus (Omicron variant) into negative clinical nasal swab matrix at 2xLoD. The studies support that the test is robust in the intended use condition with an insignificant risk of erroneous result.
VIII Proposed Labeling:
The labeling supports the finding of substantial equivalence for this device.
IX Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
