Evaluation of the clinical performance of a magnetic force-assisted electrochemical immunoassay for the detection of SARS-CoV-2 antigens.

Rapid antigen (Ag) tests for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) provide quick results, do not require specialized technical skills or infrastructure, and can be used as a point-of-care method to prevent the spread of coronavirus disease (COVID-19). The performance of a magnetic force-assisted electrochemical immunoassay-based test, namely the MARK-B COVID-19 Ag test (BBB, Sungnam, Republic of Korea), was evaluated using 170 nasopharyngeal swab specimens and compared to that of RT-PCR and commercial rapid Ag test (STANDARD Q COVID-19 Ag Test, SD Biosensor, Suwon-si, Republic of Korea). The overall sensitivity and specificity of the MARK-B test were 90.0% (95% CI 79.4%-96.2%) and 99.0% (95% CI 95.0%-99.9%), respectively, with a kappa coefficient of 0.908. The correlations between the electrical current values of MARK-B and the Ct values of RT-PCR were -0.898 (E gene, 95% CI -0.938 to -0.834) and -0.914 (RdRp gene, 95% CI -0.948 to -0.860), respectively. The limit of detection of the MARK-B was measured using the viral culture reference samples and found to be 1 x 102 pfu/mL. The magnetic force-assisted electrochemical immunoassay-based Ag test can be used to rapidly detect SARS-CoV-2 infections, and the corresponding fully automated portable device can provide easy readability and semi-quantitative results.

Introduction

Reverse-transcription polymerase chain reaction (RT-PCR) is considered the standard method for the diagnosis of coronavirus disease (COVID-19) because of its high sensitivity and specificity [1-3]. However, RT-PCR has the following disadvantages: it is expensive in terms of the cost of testing, requires established laboratory facilities, and involves a relatively longer test time [4]. Furthermore, COVID-19 testing in asymptomatic populations is increasing, which has led to an increased gap between the test demand and laboratory capacity [5]. Notably, rapid antigen tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be processed at the point of care, and the results are available within minutes. Moreover, rapid antigen testing is not dependent on advanced technical skills or infrastructure [6, 7]. Despite these advantages, rapid antigen tests are not recommended for the clinical diagnosis of COVID-19 owing to their relatively low sensitivity [8]. Recently evaluated rapid antigen tests for SARS-CoV-2 displayed a wide range of sensitivity (i.e., 17.5%–98%) with compatible specificity (i.e., 98%–100%) [6, 7, 9–17]. Nevertheless, if sufficient accuracy is obtained, then rapid antigen tests may replace RT-PCR based tests as a means for triaging or may play a role in rapid determination. Furthermore, rapid Ag tests can be easily delivered to the site, and they can be used for the mass screening of COVID-19 patients and in a more targeted manner at airports, schools, and international/regional borders [18, 19].

The MARK-B COVID-19 Ag test (MARK-B, BBB Inc., Sungnam, Republic of Korea) is a portable point-of-care device based on magnetic force-assisted electrochemical immunoassay (MESIA) designed to detect SARS-CoV-2 nucleocapsid antigens. Notably, this newly developed immunoassay, MESIA, is reportedly suitable for measuring low concentrations of proteins for cancer detection and has the potential to detect other protein biomarkers [20, 21]. MESIA may provide enhanced sensitivity for point-of-care devices that involve the use of immunoassays to detect SARS-CoV-2. Herein, we evaluated the clinical performance of the MESIA-based MARK-B test for the first time, compared to that of RT-PCR and a commercially available rapid antigen (Ag) test. Rapid Ag testing with improved accuracy may serve a variety of purposes in preventing the spread of COVID-19.

Methods

Clinical sample collection

Nasopharyngeal swab (NP) specimens were collected from patients with COVID-19 symptoms. These patients were subjected to an RT-PCR test for SARS-CoV-2 by clinicians in Eunpyeong St. Mary’s Hospital or Incheon St. Mary’s Hospital, Republic of Korea, between October 2020 and February 2021. NP specimens were placed in a universal transport medium (UTM; T-SWAB TRANSPORT CTM, Noble Biosciences, Hwaseong-si, Republic of Korea), and they were initially evaluated using RT-PCR. The remnant NP specimens were stored at −70°C, and they were used to evaluate the clinical performance of the MARK-B tests. Clinical information, such as symptoms and days from onset of symptoms (DFOS), was retrieved from medical records. This study was approved by the Institutional Review Board of the Catholic University of Korea, Republic of Korea (XC21DDDT0025), and the informed consent was waived.

MESIA for SARS-CoV-2 antigen detection

The MARK-B test is an in vitro medical device that is based on principles of MESIA and intended for the qualitative and semi-quantitative detection of the SARS-CoV-2 nucleocapsid antigens. When a sample is loaded into a cartridge, it flows into the microchannel, containing gold-coated magnetic nanoparticles and an electrochemical sensor. The analytes form immune complexes under external magnetic fields. The targeted antigen level is measured based on the electrochemical redox signal of gold-coated magnetic nanoparticles conjugated with the antibodies. The electrical signal is measured using a fully automated portable device, the MARK-B 1 Analyzer (BBB), and the result is determined in 10 min based on the cut-off value that has been set as per the manufacturer’s instructions. The preset cut-off value is determined for each lot once manufactured based on the mean and the standard deviation of the electrical signal obtained from negative samples. The QR code on each cartridge contains the information and the analyzer can recognize the cut-off of each cartridge when the QR code is scanned. The assay provides a result that indicates the absence or presence of SARS-CoV-2, along with the quantity of the captured targets from the specimen, measured using the electrical signals.

The MARK-B tests using NP specimens were performed according to the manufacturer’s instructions, which recommended the direct use of a nasopharyngeal swab or an aliquot of UTM. The previously stored UTM samples were thawed at room temperature for 30 min and vortexed for 10 s. Manufacturer recommends using 400 μL of UTM to extract SARS-CoV-2 nucleocapsid proteins from the specimen by mixing it with the extraction buffer provided with the product. However, the remnant NP specimen after RT-PCR was not enough. Therefore, the volume of the extraction buffer added was 200 μL for MARK-B™ COVID-19 Ag, and the sample was 1:1 diluted after UTM is added. Three drops of the specimen were applied to the cartridge, and the device was subjected to operation. Test results above the lot-specific cut-off value (i.e., 8.325 μA, Lot No. 0AAD0060B120131) were considered positive.

RNA extraction and real-time RT-PCR for SARS-CoV-2 detection

Viral RNA extracted from the nasopharyngeal specimens (200 μL each) was performed using the NX-48 viral NA kit (Genolution, Seoul, Republic of Korea) and the Nextractor NX-48 system (Genolution). Nucleic acids were extracted according to the manufacturer’s instructions. SARS-CoV-2 was amplified using real-time RT-PCR, with a commercial Real-Q 2019-nCoV Detection Kit (BioSewoom, Seoul, Republic of Korea). The E and RdRp genes of SARS-CoV-2 were amplified over 40 cycles using the Applied Biosystems 7500 RT-PCR system (Thermo Fisher Scientific, Waltham, MA). Samples were considered as SARS-CoV-2-positive when both targets of viral RNA had been amplified under a cycle threshold (Ct) of 38.0.

Rapid antigen test for SARS-CoV-2

The STANDARD Q COVID-19 Ag Test (SD Biosensor, Suwon-si, Republic of Korea) was used for the detection of SARS-CoV-2 antigens in NP specimens. Notably, the STANDARD Q COVID-19 Ag (SDQ) and the MARK-B tests were conducted concurrently. The stored UTM samples were thawed at room temperature for 30 min and then vortexed for 10 s. The manufacturer recommends using 350 μL of UTM and 350 μL of extraction buffer for the dilution factor 1:1. As described earlier, the remnant NP specimens was not enough, 200 μL of the NP specimen from stored UTM was mixed with the extraction buffer provided in the test kit (1:1). Three drops of the extracted specimen were applied to the test device, and the result was recorded within 15–30 min. The test result was considered positive when both the control and the test lines were colored.

Limit of detection with viral culture samples

To compare the limit of detection (LOD) between two rapid Ag kits, a serial dilution of SARS-CoV-2 samples were used. Vero E6 cells (Korean Cell Line Bank, Seoul, Korea) were cultured and incubated with the SARS-CoV-2 strain (BetaCoV/Korea/KCDC03/2020: NCCP 43326, National Culture Collection for Pathogens, Osong, Korea). The viral concentration was quantified to be equivalent to 6.5 x 105 pfu/ml and samples were serially diluted to 1.0 x 105 pfu/ml, 1 x 104 pfu/ml, 1 x 103 pfu/ml, 4 x 102 pfu/ml, 2 x 102 pfu/ml, and 1 x 102 pfu/ml. MARK-B and SDQ tests were repeated five times for each diluted sample according to the manufacturer’s instructions. Cell culture procedures were performed according to biosafety level 3 (BSL-3) conditions.

Statistical analysis

Statistical analysis was performed using the MedCalc software ver 19.6.1 (MedCalc Software, Ostend, Belgium). Figures were created using GraphPad Prism ver 9.1.2 (GraphPad Software, San Diego, CA). The Kolmogorov–Smirnov test was used to assess the normality of the distribution. The correlation between the Ct values of the E and RdRp genes obtained via RT-PCR and the measured MARK-B electrical current values was evaluated using Pearson’s correlation coefficient (r). Receiver operating characteristic (ROC) curve analysis was performed to evaluate the MARK-B lot-specific cut-off value with clinical samples. Cohen’s kappa coefficient (k) was used to assess the inter-rater reliability among SARS-CoV-2 antigen assays. Notably, k < 0 indicated no agreement, while agreement was considered to be slight for k = 0–0.20, fair for k = 0.21–0.40, moderate for k = 0.41–0.60, and substantial for k = 0.61–0.80; k = 0.81–1 represented an almost perfect agreement. The statistical significance threshold was set at P < 0.05.

Results

In total, 60 samples were analyzed using RT-PCR and were considered SARS-CoV-2-positive, while 110 samples were considered SARS-CoV-2-negative. Among the 60 samples that were confirmed as positive, 24 NP specimens had been submitted 0–3 DFOS, 24 NP specimens had been collected 4–7 DFOS, and 12 had been obtained >8 DFOS.

The comparison of the MARK-B and SDQ test results with the RT-PCR results is summarized in Table 1. Overall, MARK-B was characterized by 90.0% sensitivity (95% CI, 79.4%–96.2%) and 99.0% specificity (95% CI, 95.0%–99.9%) for SARS-CoV-2 Ag detection, and there were six false negatives. The k value of MARK-B compared to RT-PCR was 0.908. Based on the RdRp Ct value ranges (Table 2), MARK-B displayed 100% (i.e., 53/53) sensitivity for specimens characterized by Ct ≤ 30, but 14.2% (i.e., 1/7) sensitivity for specimens with Ct > 30. Furthermore, the sensitivity of the MARK-B test was estimated for RT-PCR-positive samples according to DFOS. MARK-B displayed a sensitivity of 91.6% (i.e., 22/24) for samples taken 0–3 DFOS, 91.6% (i.e., 22/24) for samples obtained 4–7 DFOS, and 83.3% (10/12) for specimens sampled ≥ 8 DFOS.

Table 1

Sensitivity and specificity of MARK-B and SDQ compared to those of real-time RT-PCR.

		Real-time RT-PCR
		Positive	Negative
MARK-B	Positive	54	1	MARK-B sensitivity = 90.0% (95% CI = 79.4%–96.2%)
	Negative	6	109	MARK-B specificity = 99.0% (95% CI = 95.0%–99.9%)
				k value = 0.908
SDQ	Positive	34	0	SDQ sensitivity = 56.7% (95% CI = 43.2%–69.4%)
	Negative	26	110	SDQ specificity = 100% (95% CI = 96.7%–100%)
				k value = 0.628

CI, confidence interval.

Table 2

Sensitivity of MARK-B and SDQ according to Ct range and target genes of real-time RT-PCR.

		E gene			RdRp gene
		< 25	25–30	> 30	< 25	25–30	> 30
MARK-B	Positive	42	11	1	40	13	1
	Negative	0	2	4	0	0	6
	Sensitivity (95% CI)	100% (91.5%–100%)	84.6% (54.5%–98.0%)	20.0% (0.5%–71.6%)	100% (91.1%–100%)	100% (75.2%–100%)	14.2% (0.3%–57.8%)
SDQ	Positive	32	2	0	33	1	0
	Negative	10	11	5	7	12	7
	Sensitivity (95% CI)	76.1% (60.5%–87.9%)	15.3% (1.9%–45.4%)	0% (0%–52.1%)	82.5% (67.2%–92.6%)	7.6% (0.1%–36.0%)	0% (0%–40.9%)

CI, confidence interval.

The mean electrical current values of MARK-B for RT-PCR-positive samples (i.e., 42.34 (SD 29.31) were significantly higher (P < 0.001, 95% CI = −42.43 to −31.4; Fig 1A) than those of RT-PCR-negative samples (i.e., 5.42 (SD 1.48)). The analysis of the correlation between the Ct values of the E gene and the electrical current values of MARK-B revealed a value of r = −0.898 (95% CI −0.938 to −0.834, P < 0.001), and that between the Ct values of the RdRp gene and the MARK-B values revealed a value of r = −0.914 (95% CI −0.948 to −0.860, P < 0.001; Fig 1B). There were six false-negative cases with MARK-B (Fig 1B), and their values were below the lot-specific cut-off value (i.e., 8.325). To evaluate the MARK-B lot-specific cut-off value with clinical samples, an ROC curve analysis was conducted. For the 170 clinical samples, the highest efficiency was estimated at a cut-off of 8.235 with an area under the curve of 0.930 (95% CI 0.880–0.963; Fig 1C). The sensitivity and specificity of the MARK-B test with a clinically estimated cut-off (8.235) yielded the same results as when the lot-specific cut-off value was applied.

Fig 1

RT-PCR and MARK-B test results.

(A) Peak current values of RT-PCR-positive and RT-PCR-negative samples. (B) Correlation between peak current values (MARK-B, log2 scale) and the Ct values of the RdRp gene (60 RT-PCR-positive samples; ○, MARK-B false negative). (C) Receiver operating characteristic (ROC) curve analysis. The MARK-B test indicated an area under the ROC curve value of 0.930 (95% CI 0.880–0.963). Dotted line: lot-specific cut-off (8.325 μA), ****P < 0.0001.

Overall, the SDQ test was characterized by 56.7% sensitivity (95% CI 43.2%–69.4%) and 100% specificity (95% CI 96.7%–100%) for SARS-CoV-2 Ag detection. There were 26 false negatives, and the k value was 0.628 for SDQ and RT-PCR tests (Table 1). The sensitivity of the SDQ test decreased markedly for specimens with Ct ≥ 25 and showed 0% sensitivity for specimens with Ct > 30. There were 10 cases of weak positives with SDQ tests. Notably, MARK-B yielded positive results for the samples corresponding to the 10 weak positive cases. The mean Ct value of the RdRp gene of MARK-B-positive/SDQ-positive samples was 18.5 (SD 2.8), that of MARK-B-positive/SDQ-weak positive was 22.4 (SD 1.6), that of MARK-B-positive/SDQ-negative was 26.4 (SD 2.5), and that of MARK-B-negative/SDQ-negative was 31.8 (SD1.1) (Fig 2). There were significant differences between the mean Ct values of the RdRp gene in each of these four groups (Fig 2).

Fig 2

MARK-B and SDQ results compared to the Ct values (RdRp gene) of RT-PCR-positive samples.

MB: MARK-B COVID-19 Ag test, SDQ: STANDARD Q COVID-19 Ag, +: positive, -: negative, ***P < 0.001, ****P < 0.0001.

The LOD of the two rapid kit was tested with reference viral culture samples. Cultured viral samples were diluted to six concentrations. The measured LOD of MARK-B was 1 x 102 pfu/mL, and SDQ was 1.0 x 104 pfu/mL, respectively (Table 3).

Table 3

Comparison of the limit of detection for MARK-B and SDQ with cultured viral samples.

		Virus concentration (pfu/mL)
		Negative	1 x 102	2 x 102	4 x 102	1 x 103	1 x 104	1 x 105
MARK-B	Mean (μA)	6.70	8.782	9.615	10.727	18.917	39.896	61.816
	SD	0.67	0.24	0.49	1.03	2.11	2.41	5.37
SDQ		Negative	Negative	Negative	Negative	Negative	Weak positive	Positive

Discussion

In the present study, the performance of the MESIA-based Ag test MARK-B was evaluated using clinical samples and compared with the performance of RT-PCR and commercial Ag tests. The MARK-B tests displayed 90.0% sensitivity and 99.0% specificity across the samples. Moreover, the MARK-B test exhibited 100% sensitivity for samples whose RT-PCR results were Ct < 25 and 84.6% sensitivity for both E and RdRp genes (Ct = 25–30). Additionally, it was characterized by 95.9% overall agreement and a 0.908 k value, indicating an extremely good match between the results of RT-PCR and the MARK-B test. Both the commercial and the MARK-B Ag tests exhibited higher sensitivity for Ct values <25, and the sensitivity decreased as Ct values increased. The SDQ test reportedly exhibits a sensitivity above 95% for Ct values <25 and 53.9–62.1% for Ct value >25 [7, 22–25]. In the present study, SDQ tests displayed 76.1% sensitivity for Ct values <25 and declined sharply for Ct value >25. This study was conducted using remnant specimens; therefore, the storage status of the samples and the use of UTM might have affected the sensitivity of the rapid tests. In general, studies that involved the use of UTM reported lower sensitivity than studies that involved the use of NP specimens [7, 13, 22, 24, 25]. All RT-PCR-positive samples were maintained under freezing conditions at −70°C before Ag tests, and few samples were refrozen after subjection to thawing once. Notably, viral accessibility can be influenced by specimen storage conditions such as the freeze-thaw cycle. One freeze-thaw cycle increased the Ct value to 0.41, and two cycles of freezing and thawing increased the Ct value to 0.82 [26]; thus, more freeze-thaw cycles tended to increase the Ct value [27].

The MARK-B tests are automatically conducted using a portable device analyzer that regulates the magnetic field and measures electrochemical signals. The qualitative results were determined based on a cut-off value, and the measured electrochemical signals were also presented. The ROC curve analysis with clinical samples demonstrated that the lot-specific cut-off value was appropriately established. The use of visual readout rapid Ag tests can produce ambiguous results at low antigen concentrations, while instrument-based Ag tests yield results with clarity. For instance, when interpreting the visual readout Ag test, the presence of a line, regardless of the faintness of the line, indicates a positive result. There were 10 weak-positive cases within the SDQ results and 20 cases of MARK-B-positive/SDQ-negative results, and there were significant differences between these sample groups. Notably, the MARK-B device provided readability for clinical samples with higher Ct values, and the MESIA technique improved the sensitivity of the immunoassay for detecting SARS-CoV-2 antigens. The LOD measurement of two rapid Ag tests were conducted to verify the sensitivity differences between the rapid Ag tests using viral culture reference samples. In line with tests using clinical samples, MARK-B tests showed a LOD at approximately 102 times lower viral concentrations than SDQ.

Furthermore, the electrical current values of MARK-B and the Ct values of RT-PCR were highly correlated. We plotted log2(current) values vs. Ct values and fitted with a line. While the signal was saturated at the concentrations higher than Ct 20, the log2(current) vs. Ct showed a linear relationship in the range from Ct 20 to Ct 33, as shown Fig 1B. These results indicate that the electrochemical signals measured with the MESIA are proportional to the concentration of antigens, which suggests that MESIA can help provide reliable semi-quantitative results in conditions where RT-PCR is not available.

In conclusion, the MARK-B test, a MESIA-based rapid Ag test, showed higher sensitivity compared to commercial rapid Ag tests for the detection of SARS-CoV-2. Furthermore, the MESIA technique and automated portable device provided results with improved clarity in 15 min as well as reliable semi-quantitative measurement. These results indicate that these rapid Ag tests can be useful for preventing the spread of COVID-19 via timely diagnosis and subsequent containment measures.

3 Aug 2021

PONE-D-21-20548

Evaluation of the clinical performance of a magnetic force-assisted electrochemical immunoassay for the detection of SARS-CoV-2 antigens

PLOS ONE

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Reviewer #1: The Authors evaluated the MARK-B COVID-19 Ag test (MARK-B, BBB, Sungnam, Republic of Korea) that is a portable point-of-care device based on the use of a magnetic force-assisted electrochemical immunoassay (MESIA) designed to detect SARS-CoV-2 antigens. Notably, this newly developed immunoassay, MESIA, reportedly suitable for measuring low concentrations of proteins for cancer detection and with the potential to detect any other biomarkers.

The evaluation was done against RT PCR as the gold standard for SARS COV-2 detection and another immunochromatographic manual antigen assay for SARS CoV-2, showing a significant higher correlation with RT PCR and a significant higher sensitivity over the standard antigen assay. Overall, this data confirms that MESIA may provide enhanced sensitivity for point-of-care devices that involve the detection of SARS-CoV-2.

The study is well conducted, each section is well written and developed, statistical measures correctly applied. There is one general limitation in this study:

The MARK-B test has not been CE/FDA-cleared or approved so far for SARS CoV-2, but these validations are pending. Therefore, this is a clear limitation of the study conducted by the Authors, in terms of extension and commercialization of this device. The Authors should discuss this issue and clarify if there are any on-going news on this important issue which is essential for the scientific community. Moreover, the test is not cited by FIND as the date of today July 15 among the 653 antigen immunoassays for SARS CoV-2 enlisted in the web site https://www.finddx.org/covid-19/pipeline/?section=immunoassays#diag_tab

Is this just for Asia? The Authors must clarify it.

Minor issues

Material and Methods:

The semi-quantitative/quantitation system used by MESIA for their electric signal should be specified by the Authors as they use it in the Figures, as well as the nature of the cut-off value and the type of antigen detected by this system.

Reviewer #2: This paper describes the evaluation of a magnetic force-assisted electrochemical immunoassay -based test compared to other commercial rapid Ag tests by application to clinical samples. Authors have successfully compared the results provided by this device with those from other methodologies applying them to a large number of samples. In my opinion, the information offered in the article is very interesting, the electrochemical sensor has demonstrated a good behavior, but the experimental work and the new data in the article are not relevant from the point of view of scientific research. I consider that the paper should not be published in its current state in PLOs One.

Reviewer #3: This is a clear well-written paper describing the performance of a new Ag test for diagnosis of COVID-19 infection. As an independent of assessment of the test, the publication provides important data to users who may be considering use of the test. I recommend publication after revision to address some minor weaknesses in the paper:

1. The main weakness is associated with the comparison to an existing commercial Ag test and the interpretation of the comparison

a. In the abstract and main text their are locations where the authors describe their paper as comparing their test to commercial antigen tests. These statements should be clarified to indicate that they compared to a (as in one) antigen test, and isn't necessarily reflective of how the test would perform to the range of available tests.

b. Some discussion is warranted regarding the criteria for the selection of the comparator test, and the relative performance of this test. Relative to the many Ag tests that are now available commercially, does the published data for the SDQ test place it at the top, middle or bottom of the range for clinical sensitivity and accuracy?

c. It would be helpful to provide some testing with a reference sample (for example, one of the available viral culture reference samples or a sample of recombinant nucleocapsid protein) that could allow direct comparison of the analytical performance of the antigen assays to each other (and potentially other commercial tests) and determination of whether the clinical performance is a direct reflection of the analytical performance).

2. The volume of UTM used to extract the sample should be provided and, if applicable, a discussion of how the volume of UTM may differ from the volume of extraction buffer used in the standard swab protocols for the MESIA and SDQ tests, and how that may affect performance.

3. Ct should be proportional to Log2(RNA concentration) since it represents the number of RNA doubling steps required to achieve a detectable PCR signal. It is therefore probably most appropriate to plot Log2(MESIA current) vs. Ct, which should provide a slope approaching 1. If the log(current) vs. Ct is not linear, that should be discussed.

4. In the methods section, it isn't very clear how the MESIA technique works. I think readers would provide a little more detail or, alternatively, a reference to where they could go for more details.

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Reviewer #1: Yes: Valeria Ghisetti, MD, Director of the laboratory of Microbiology and Virology, Amedeo di Savoia Hospital, Turin, Italy

Reviewer #2: No

Reviewer #3: No

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13 Sep 2021

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27 Sep 2021

Evaluation of the clinical performance of a magnetic force-assisted electrochemical immunoassay for the detection of SARS-CoV-2 antigens

PONE-D-21-20548R1

Dear Dr. Lee,

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Reviewer #3: All comments have been addressed

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Reviewer #3: Yes

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Reviewer #3: Yes

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Reviewer #3: Yes

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Reviewer #3: Yes

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Reviewer #3: Some minor comments/suggestions based on the response to my original review:

1. The description of the assay technology in your response to the reviewers comments is much clearer than in the edits to the manuscript. I suggest just using the text from the response in the manuscript.

2. The line in the log2 antigen vs. Ct count plot should be calculated using the log2 transformed values and should look like a straight line in the plot. Interestingly, the slope of that line will be about -0.3 indicating that an 8-fold change in nucleic acid levels (3 Ct counts) corresponds to roughly a 2-fold change in the antigen assay signal (roughly a cube root dependence). The table of assay signals for the virus titration also shows roughly a cube root dependence.

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Reviewer #3: No

29 Sep 2021

PONE-D-21-20548R1

Evaluation of the clinical performance of a magnetic force-assisted electrochemical immunoassay for the detection of SARS-CoV-2 antigens

Dear Dr. Lee:

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