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. 2021 Mar 4;16(3):e0247711. doi: 10.1371/journal.pone.0247711

Validation of a new automated chemiluminescent anti-SARS-CoV-2 IgM and IgG antibody assay system detecting both N and S proteins in Japan

Rin Yokoyama 1, Makoto Kurano 1,2,*, Yoshifumi Morita 1, Takuya Shimura 1, Yuki Nakano 1, Chungen Qian 3, Fuzhen Xia 4, Fan He 4, Yoshiro Kishi 5, Jun Okada 5, Naoyuki Yoshikawa 1, Yutaka Nagura 6, Hitoshi Okazaki 6, Kyoji Moriya 7, Yasuyuki Seto 8, Tatsuhiko Kodama 9, Yutaka Yatomi 1,2,*
Editor: Katerina Kourentzi10
PMCID: PMC7932516  PMID: 33661990

Abstract

PCR methods are presently the standard for the diagnosis of Coronavirus disease 2019 (COVID-19), but additional methodologies are needed to complement PCR methods, which have some limitations. Here, we validated and investigated the usefulness of measuring serum antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using the iFlash3000 CLIA analyzer. We measured IgM and IgG titers against SARS-CoV-2 in sera collected from 26 PCR-positive COVID-19 patients, 53 COVID-19-suspected but PCR-negative patients, and 20 and 100 randomly selected non-COVID-19 patients who visited our hospital in 2020 and 2017, respectively. The repeatability and within-laboratory precision were obviously good in validations, following to the CLSI document EP15-A3. Linearity was also considered good between 0.6 AU/mL and 112.7 AU/mL for SARS-CoV-2 IgM and between 3.2 AU/mL and 55.3 AU/mL for SARS-CoV-2 IgG, while the linearity curves plateaued above the upper measurement range. We also confirmed that the seroconversion and no-antibody titers were over the cutoff values in all 100 serum samples collected in 2017. These results indicate that this measurement system successfully detects SARS-CoV-2 IgM/IgG. We observed four false-positive cases in the IgM assay and no false-positive cases in the IgG assay when 111 serum samples known to contain autoantibodies were evaluated. The concordance rates of the antibody test with the PCR test were 98.1% for SARS-CoV-2 IgM and 100% for IgG among PCR-negative cases and 30.8% for SARS-CoV-2 IgM and 73.1% for SARS-CoV-2 IgG among PCR-positive cases. In conclusion, the performance of this new automated method for detecting antibody against both N and S proteins of SARS-CoV-2 is sufficient for use in laboratory testing.

Introduction

In December 2019, the first pneumonia cases caused by an unknown microorganism were identified in Wuhan, China [1]. The pathogen was identified as a novel betacoronavirus and was named “severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)” [2]. SARS-CoV-2 is phylogenetically similar to SARS-CoV, which caused outbreaks of a severe respiratory syndrome in China in 2002 [3]. The symptoms of coronavirus disease 2019 (COVID-19), which is the respiratory syndrome caused by SARS-CoV-2, are fever, cough and lymphopenia [4]. Chest computed tomography examinations of COVID-19 patients are characterized by the bilateral distribution of patchy shadows or ground-glass opacities [5]. Since early December 2019 and as of June 15, 2020, over 7,800,000 cases of COVID-19 have been confirmed and 430,000 deaths have been reported throughout the world [6], and the World Health Organization has reported a fatality rate for cases defined as pneumonia of approximately 2% [7].

Currently, COVID-19 is diagnosed by the clinical presentation of the patient, as described above, and the detection of SARS-CoV-2 RNA in respiratory specimens, such as a nasal swab or sputum, using real-time reverse-transcription polymerase chain reaction (RT-PCR) [8, 9]. However, this method requires skilled technicians who know how to handle genetic samples and perform PCR tests and occasionally causes false-negative results because of the viral replication window, a low viral titer, or incorrect sample collection [10]. Moreover, the sampling of respiratory specimens exposes medical staff to a higher risk of secondary infection through aerosolization than the sampling of sera [11, 12]. Therefore, other methods are required to complement PCR testing.

Candidate complementary tests include both antigen and antibody tests. Regarding antigen tests, although this method does not require special skills for performing genetic testing, there remains a high risk of secondary infection during sampling, and the sensitivity of antigen tests is reportedly lower than that of PCR testing [13, 14]. Antibody tests are another candidate. Compared with PCR testing, this serological test method has a faster turn-around time and requires easier and safer sample collection and less specialized skills for technicians; furthermore, when we interpret the results of an antibody testing considering the duration after the onset of COVID-19, this test would give us important information in diagnosing COVID-19. The main concern regarding antibody tests is the high frequency of false-positive cases, which is a parameter that should depend on the quality of the test product [15, 16]. Recently, Shenzhen YHLO Biotech Co., Ltd (China) has developed an antibody test with a high specificity [17–19]; however, this method has not yet been validated in the Japanese population. Therefore, in the present study, we aimed to validate the measurement of IgM and IgG antibodies against SARS-CoV-2 in sera and to investigate the usefulness of this method for the diagnosis of COVID-19.

Materials and methods

Subjects

We enrolled a total of 26 COVID-19-positive cases and 53 COVID-29-suspected cases who were hospitalized at The University of Tokyo Hospital. Confirmed COVID-19 cases were defined based on the guidelines of The University of Tokyo Hospital. Briefly, patients with acute respiratory infection syndrome accompanied by detectable SARS-CoV-2 RNA in a throat swab or sputum at least once were confirmed as having COVID-19 (PCR-positive cases). Suspected patients were defined as subjects with respiratory symptoms, a history of overseas travel, or a high-risk contact with a confirmed COVID-19 case but with negative PCR results. We received the patient’s whole blood in the collection tube coated with silica and thrombin for the clinical laboratory testing. Then, the serum was separated by centrifuging at 2,300 g for 5 minutes and carried out clinical testing. We collected residual sera available after routine clinical testing and kept it frozen at -20°C until measurement. The serum samples before the onset of the infection were collected by chance since all serum samples investigated in our laboratory were stored for 3 weeks from the day when the routine laboratory testing was performed. Three of the subjects of whom we collected the serum samples before the onset of COVID-19 symptoms had been confirmed SARS-CoV-2 PCR negative. In other four subjects and control cases collected in March 2020, symptoms of cold were not described in medical records. We enrolled the COVID-19 subjects and the suspicious subjects, which we can collect residual serum samples between April 22, 2020 and June 22, 2020. The serological tests were performed using the sample that had been collected on the day closest to the day on which the sample for the PCR test had been collected. The mean days (±S.D.) between the antibody test and the onset of the symptom or the PCR test were 11.3 (±6.70) or 5.67 (±5.67) days, respectively. As control groups, we randomly selected 20 and 100 outpatients who had visited The University of Tokyo Hospital in March 2020 or January-December 2017 and were not complicated with autoimmune diseases, respectively. We also collected 111 serum samples known to contain autoantibodies. All samples used in this study were stored biological samples and were de-identified before we accessed them.

The current study was performed in accordance with the Declaration of Helsinki. Informed consent was obtained in the form of an opt-out form on the institution’s website. The institutional ethics committee approved this informed consent plan. This study was conducted with the approval of The University of Tokyo Medical Research Center Ethics Committee (2019300NI-3).

Antibody testing

Antibody testing was performed using SARS-CoV-2 IgM and IgG chemiluminescence immunoassay (CLIA) kits supplied by Shenzhen YHLO Biotech Co., Ltd. (China) and an iFlash3000 fully automated CLIA analyzer also from Shenzhen YHLO Biotech Co., Ltd. (China). Two antigens of SARS-CoV-2 (nucleocapsid protein [N protein] and spike protein [S protein]) were coated on the magnetic beads of these CLIA assays. The assay procedure was described by Qian C, et al [20]. Briefly, the acridinium-labeled anti-human IgM or IgG conjugate antibody was used to detect the antibody bound to the beads. The SARS-CoV-2 IgM or IgG titers in 5 uL of the sample were calculated as relative light units (RLU) obtained from the analyzer and were described as arbitrary units per milliliter (AU/mL) by comparing the RLU detected by iFlash optical system with the cutoff calculated from the SARS-CoV-2 IgM or IgG calibrators containing anti-SARS-CoV-2 IgM or IgG chimeric antibody. According to the manufacturer’s instructions, the cutoff value for a positive SARS-CoV-2 IgM/IgG result was deemed as 10 AU/mL. To measure the antibody titers against a single antigen, we used the magnetic beads coated with either antigen. If the SARS-CoV-2 IgG titer was over 40 AU/mL, the sample was diluted with non-reactive serum and the antibody titers were measured once again.

To compare with an established antibody test against SARS-CoV-2, we measured 385 serum samples collected from COVID-19 positive or suspected patients with an anti-SARS-CoV-2 electrochemiluminescence immunoassay (ECLIA) kit obtained from Roche diagnostics K.K. (Japan), equipped with cobas e 601 manufactured by Roche diagnostics K.K. (Japan). According to the manufacturer’s instructions, the cutoff value for a positive SARS-CoV-2 antibody result was deemed as 1 COI (cutoff index).

Method validation

We evaluated the assay precision, according to the guideline of The Clinical & Laboratory Standards Institute (CLSI) documents EP15-A3 [21]. We investigated the assay precision for five days and five replicates per run, using two manufacturer’s controls as a sample. We compared the repeatability and within-laboratory precision, using the results obtained in this study and the upper verification limits calculated from the precision values proposed by Qian C et al, which were obtained by measuring three to four serum samples in duplicates for each run and two runs per day over 20 testing days, following CLSI documents EP5-A3 [20]. Linearity was investigated using two kinds of pooled serum samples. Briefly, each sample was diluted with pooled non-reactive serum in two-fold serial dilutions for ten times. Additionally, linearity study was also performed according to the CLSI document EP06 [22]. The range which any nonlinear coefficients were not significant when a least-squared regression using polynomials of 1st, 2nd and 3rd order was regarded as a linear range. To investigate the existence of the prozone phenomenon, we diluted the samples with high concentrations of SARS-CoV-2 IgM and IgG titers using ten-fold serial dilutions for the SARS-CoV-2 IgM assay and two-fold serial dilutions for the SARS-CoV-2 IgG assay.

To evaluate the detection capability, we performed the verification of limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ), according to the CLSI document EP17-A2 [23]. The LoD was determined with the proportions of false positives (α) less than 5% and false negatives (β) less than 5%. Non-reactive serum was used as a blank sample. To evaluate the LoQ, we measured 8 samples for SARS-CoV-2 IgM and 10 samples for SARS-Cov-2 IgG with 6 replicates, respectively. The LoQ was determined as the minimum concentration no more than 10% of CV. We performed method validation using a single reagent lot.

Statistical analysis

For the precision study, analysis of variance (ANOVA) was used to estimate the repeatability and within-laboratory precision. For linearity study, whether a nonlinear coefficient was significantly different from zero was evaluated by t-test. These data were analyzed using JMP software (North Carolina, USA). The data evaluating the cross-reactivity were analyzed using StatFlex software (Osaka, Japan). The results were expressed as the mean ± SD. The Dunn test was used for comparisons of antibody titers between the control and other groups. A value of p < 0.05 was regarded as statistically significant in all the analyses.

Results

Precision and accuracy of antibody testing

First, we performed the verification of repeatability and within-laboratory precision according to the CLSI document EP15-A3 for five days and with five replicates per run, using two manufacturer’s controls each assay (Table 1). The repeatability of SARS-CoV-2 IgM was ranged from 1.90% to 2.13%, and the within-laboratory precision was from 2.48% to 4.08%. The repeatability of SARS-CoV-2 IgG was ranged from 1.03% to 1.65%, and the within-laboratory precision is from 1.87% to 2.17% (Table 1). Those precision values were lower than the upper verification limits (from 2.80% to 4.32 for the repeatability of SARS-CoV-2 IgM, from 3.02% to 4.08% for the within-laboratory precision of SARS-CoV-2 IgM, from 3.11 to 4.30% for the repeatability of SARS-CoV-2 IgG, and from 3.12 to 5.13% for the within-laboratory precision of SARS-CoV-2 IgG), which were calculated from the precision values reported by Qian C et al [20].

Table 1. Repeatability and within-laboratory precision of SARS-CoV-2 IgM/IgG according to CLSI EP15-A3.

Repeatability Within-laboratory precision
Sample Mean (AU/mL) SD CV (%) SD CV (%)
SARS-CoV-2 IgM 1 5.4 0.12 2.13 0.22 4.08
2 25.2 0.48 1.90 0.63 2.48
SARS-CoV-2 IgG 3 5.5 0.09 1.65 0.10 1.87
4 19.6 0.20 1.03 0.43 2.17
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We measured SARS-CoV-2 IgM or IgG in two manufacturer’s controls for 5 days, 5 replicates per run, following to the CLSI document EP15-A3. Repeatability and within-laboratory precision were calculated using ANOVA.

Measurement range of SARS-CoV-2 antibody testing

To explore the measurement range of this antibody test, we performed a linear regression analysis. When we investigated linearity using samples with moderate antibody titers, the curves showed a good linearity between 0.6 AU/mL and 112.7 AU/mL for SARS-CoV-2 IgM and between 3.2 AU/mL and 55.3 AU/mL for SARS-CoV-2 IgG (Fig 1A–1D). Additionally, none of nonlinear coefficients were significant between 1.50 AU/mL and 15.92 AU/mL for SARS-CoV-2 IgM (p = 0.66 for 2nd order regression, p = 0.50 and p = 0.47 for 3rd order regression) and between 2.36 AU/mL and 18.30 AU/mL for SARS-CoV-2 IgG (p = 0.27 for 2nd order regression, p = 0.13 and p = 0.10 for 3rd order regression) (Fig 1E and 1F). Next, we measured samples with high antibody titers to determine the upper limit of the measurement range. In the SARS-CoV-2 IgM assay, the upper curve increased up to a value of 2,405 AU/mL and then reached a plateau at higher concentrations. In the SARS-CoV-2 IgG assay, the curve reached a plateau at values over 73 AU/mL (Fig 2A and 2B). When we used saline as a diluent, we observed a hook effect (Fig 2C), whereas when we used non-reactive serum, we did not observe a hook effect as shown in Fig 2A and 2B. Therefore, we diluted the samples over 40 AU/mL for SARS-CoV-2 IgG with non-reactive serum.

Fig 1. Linearity analyses of SARS-CoV-2 antibody titer.

Fig 1

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The dilution linearities of SARS-CoV-2 IgM (A, B) and SARS-CoV-2 IgG (C, D) were investigated. A sample was diluted with non-reactive serum in 5 to 8 steps; each sample was then analyzed with two replicates. The linearity studies for SARS-CoV-2 IgM (E) and SARS-CoV-2 IgG (F) were performed according to CLSI EP06-A. The sample of high antibody titer was diluted with the sample of low antibody titer in 6 to 9 steps; each sample was the analyzed with two steps.

Fig 2. Prozone phenomena and a hook effect of SARS-CoV-2 antibody titer.

Fig 2

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The prozone phenomena of SARS-CoV-2 IgM (A) and SARS-CoV-2 IgG (B) were investigated. We diluted two serum samples from infected patients with non-reactive serum in 10 steps; each sample was then analyzed with two replicates. We diluted a serum sample with a saline in 10 steps and measured SARS-CoV-2 IgG titer (C); each sample was then analyzed with two replicates.

Detection capability of SARS-CoV-2 antibody testing

To evaluate the detection capacity for SARS-CoV-2 IgM/IgG, we determined the LoB and LoD. The LoD for SARS-CoV-2 IgM was 0.74 AU/mL, determined by 130 measurements with 60 blank and 70 low level replicates, and the LoB was 0.63 AU/mL. The LoD for SARS-CoV-2 IgG was 0.53 AU/mL, determined by 120 measurements with 60 blank and 60 low level replicates, and the LoB was 0.47 AU/mL. We also investigated the LoQ. We measured 7 samples of low antibody levels for SARS-CoV-2 IgM and 10 samples for SARS-CoV-2 IgG. In both assays, CVs of the samples which were lower than the LoD showed no more than 10%. Therefore, the LoQ for SARS-CoV-2 IgM was determined as 0.74 AU/mL and that for SARS-CoV-2 IgG was 0.53 AU/mL.

Successful detection of SARS-CoV-2 IgM/IgG

To confirm that this antibody measurement system could detect SARS-CoV-2 IgM/IgG successfully, we measured the antibody titers in sera obtained before and after infection with SARS-CoV-2 in three cases of COVID-19 confirmed using RT-PCR tests. As shown in Fig 3, SARS-CoV-2 IgM and IgG were not detected before symptom onset; at several days after symptom onset, tests for both antibodies became positive and the titers gradually increased. In case 4, 6 and 7, SARS-CoV-2 IgM titers gradually increased although the titers did not increase above the cutoff value in the period of observation. In case 1 and 3, the IgM test became negative again at day 19 and day 22, respectively.

Fig 3. Time course of serum antibody titers in COVID-19 subjects.

Fig 3

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The time courses of the SARS-CoV-2 IgM and SARS-CoV-2 IgG titers in sera collected before and after the onset of COVID-19 were examined in seven patients.

Second, we obtained 100 random serum samples collected from outpatients who had visited The University of Tokyo Hospital in 2017, when SARS-CoV-2 did not exist. None of these samples had an antibody titer over 10 AU/mL, suggesting that this measurement system can detect SARS-CoV-2 IgM/IgG without false-positive results (Fig 4).

Fig 4. Serum antibody titers in sera from 2017.

Fig 4

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The SARS-CoV-2 IgM/IgG titers of sera collected from subjects (n = 100) in 2017 were measured.

Cross-reactivity with autoantibodies

Since the presence of autoantibodies can sometimes affect the results of serological tests, we measured SARS-CoV-2 IgM/IgG in residual serum samples collected from patients with one of 5 different autoimmune diseases. For IgM, most of the serum samples from the patients with autoimmune diseases did not have a result over 10 AU/mL. However, two rheumatoid factor-positive patients, one anti-double-strand DNA antibody-positive patient, and one anti-mitochondrial M2 antibody-positive patient had values that exceeded the cutoff value (Fig 5A). For IgG, none of the autoantibody-positive serum samples had a result that was over 10 AU/mL (Fig 5B).

Fig 5. Interference from autoantibodies in SARS-CoV-2 IgM/IgG assay.

Fig 5

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We collected sera from patients with autoimmune diseases and measured the SARS-CoV-2 IgM (A) and IgG (B) titers. *p < 0.05, **p < 0.01. Control, randomly selected outpatients who visited the hospital in 2020 (n = 20); RF, rheumatoid factor-positive group (n = 25); dsDNA, anti-double-strand DNA antibody-positive group (n = 26); M2, anti-mitochondrial M2 antibody-positive group (n = 20); P-ANCA, myeloperoxidase antineutrophil cytoplasmic antibody-positive group (n = 20); SS-A, anti-Sjögren’s syndrome A antibody-positive group (n = 20); Suspected, suspected COVID-19 group (n = 53); Positive, COVID-19-positive group (n = 26).

Concordance rate with PCR testing or Roche’s ECLIA kit

To investigate clinical usefulness, we compared the results of the serological antibody tests with those of PCR tests. Among the 26 PCR-positive COVID-19 cases, 8 cases (30.8%) had IgM-positive results and 19 cases (73.1%) had IgG-positive results. Among the 53 PCR-negative COVID-19-suspected cases, 52 cases (98.1%) had IgM levels below 10 AU/mL and all the cases (100%) had IgG levels below 10 AU/mL (Table 2).

Table 2. Concordance rate between the results of PCR testing and SARS-CoV-2 IgM or IgG serological testing.
SARS-CoV-2 PCR test
positive cases (n = 26) negative cases (n = 53)
SARS-CoV-2 IgM >10 AU/mL 8 (30.8%) 1
≤10 AU/mL 18 52 (98.1%)
SARS-CoV-2 PCR test
positive cases (n = 26) negative cases (n = 53)
SARS-CoV-2 IgG >10 AU/mL 19 (73.1%) 0
≤10 AU/mL 7 53 (100%)
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We measured SARS-CoV-2 IgM and IgG in PCR-positive subjects (n = 26) and PCR-negative subjects (n = 53). An antibody titer above 10 AU/mL was regarded as positive, according to the manufacturer’s cutoff.

We also compared these results of the SARS-CoV-2 IgM or IgG tests with those obtained using the Roche’s ECLIA kit. Among the 13 serum samples above 1 COI by the Roche’s kit, 4 samples (30.8%) had the SARS-CoV-2 IgM-positive results and 12 samples (92.3%) had the SARS-CoV-2 IgG-positive results. Among the 48 serum samples under 1 COI by the Roche’s kit, 46 samples (95.8%) had the SARS-CoV-2 IgM or IgG levels below 10 AU/mL, when we analyzed only the samples identical to the samples in Table 2 (Table 3A). Among the 196 serum samples above 1 COI by the Roche’s kit, 114 samples (58.2%) had the SARS-CoV-2 IgM-positive results and 193 samples (98.5%) had the SARS-CoV-2 IgG-positive results. Among the 189 serum samples under 1 COI, 179 samples (94.7%) had the SARS-CoV-2 IgM levels below 10 AU/mL and 165 samples (87.3%) had the SARS-CoV-2 IgG levels below 10 AU/mL, when we analyzed all the serum samples obtained additionally (Table 3B).

Table 3. Concordance rate between the results of the Roche’s ECLIA kit and those of the SARS-CoV-2 IgM or IgG CLIA testing.
(A)
Roche’s ECLIA kit
≥1 COI (n = 13) <1 COI (n = 48)
SARS-CoV-2 IgM ≥10 AU/mL 4 (30.8%) 2
<10 AU/mL 9 46 (95.8%)
Roche’s ECLIA kit
≥1 COI (n = 13) <1 COI (n = 48)
SARS-CoV-2 IgG ≥10 AU/mL 12 (92.3%) 2
<10 AU/mL 1 46 (95.8%)
(B)
Roche’s ECLIA kit
≥1 COI (n = 196) <1 COI (n = 189)
SARS-CoV-2 IgM ≥10 AU/mL 114 (58.2%) 10
<10 AU/mL 82 179 (94.7%)
Roche’s ECLIA kit
≥1 COI (n = 196) <1 COI (n = 189)
SARS-CoV-2 IgG ≥10 AU/mL 193 (98.5%) 24
<10 AU/mL 3 165 (87.3%)
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We investigated the concordance rate between the SARS-CoV-2 IgM or IgG and the Roche’s ECLIA kit in 61 serum samples used in Table 2 (A) and all of 385 serum samples collected from COVID-19-positive or -suspected patients (B). For the Roche’s ECLIA kit, the SARS-CoV-2 antibody titer above 1 COI was regarded as positive, according to the manufacturer’s cutoff.

Suspected false-positive IgM results might be caused by reactivity to N protein

In this study, we observed 5 suspected false-positive IgM results. As described in the Materials and Methods section, the measurement system tests the reactivity to both the N protein and the S protein of SARS-CoV-2. We investigated the reactivity of the samples to N protein and S protein separately, and only reactivity to N protein was observed in the 5 suspected false-positive samples (Table 4).

Table 4. IgM reactivity to N protein and S protein in subjects with suspected false-negative IgM results.
Sample S+N protein (AU/mL) N protein (AU/mL) S protein (AU/mL)
1 53.76 97.17 0.34
2 16.43 18.14 0.96
3 13.03 10.51 0.34
4 17.94 29.31 0.69
5 110.11 130.57 1.15
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We investigated the IgM reactivity to N protein and S protein in five subjects with suspected false-negative IgM results. N or S protein means the value measured using magnetic beads coated with either antigen, respectively.

Discussion

In this study, we validated a method for quantifying SARS-CoV-2 IgM and IgG using the iFlash3000 automated CLIA analyzer. First, the repeatability and within-laboratory precision of the SARS-CoV-2 IgM and IgG assays were obviously good in validations, following to the CLSI document EP15-A3 (Table 1). These results suggest that relatively stable data were provided by this measurement system, while some anti-SARS-CoV-2 antibody detection kits authorized for emergency use by the Food and Drug Administration (FDA) in the USA have a CV of more than 5% for intermediate precision and some manufacturers do not even publish precision data for their products. The linearity was good up to values of 112.7 AU/mL for SARS-CoV-2 IgM and 55.3 AU/mL for IgG, while the assay signal gradually reached a plateau at over 2,405 AU/mL for SARS-CoV-2 IgM and over 73 AU/mL for IgG (Fig 2). Therefore, the sample should be diluted if the measured value is expected to be higher than the measurement range. To dilute samples, non-reactive serum should be used as a diluent, since a hook effect was observed when saline is used as a diluent, especially for IgG measurements (Fig 2C). Clinical decisions are rarely affected by this phenomenon, since the measurement range that was validated in the present study covers much higher values than the manufacturer’s cutoff. Additionally, it would not be necessary to consider false negative results of SARS-CoV-2 IgG due to the extremely high titers, since the IgG titers of any participants who admitted to The University of Tokyo Hospital for COVID-19 had never become negative for SARS-CoV-2 IgG testing during the hospitalization once the seroconversion of IgG was observed. In case, the simultaneous measurement of IgM would help us rule out the possible false negativity in IgG test. To evaluate usefulness of these kits, we investigated the concordance rate between the results of the present study and those we obtained when we measured the same serum samples with Roche’s ECLIA kit, as an approved method for the SARS-CoV-2 antibody testing. The concordance rate between SARS-CoV-2 IgM/IgG and Roche’s kit were more than 90% except for the positive concordance rate between SARS-CoV-2 IgM and Roche’s kit, when we analyzed only the same samples as described on Table 2 (Table 3A). The negative concordance rate between Roche’s kit and SARS-CoV-2 IgM was 94.7% and the positive concordance rate between Roche’s kit and SARS-CoV-2 IgG was 98.5%, when we compared using all the serum samples (Table 3B).

Regarding the samples showing the discrepant results between Roche’s kit and SARS-CoV-2 IgG, three samples of which the antibody titers were below 10 AU/mL by SARS-CoV-2 IgG assay and above 1 COI by Roche’s kit were PCR-positive subjects, while we also observed seroconversion by SARS-CoV-2 IgG assay in the samples collected from the three subjects after 2 days. The antibody titers of 24 samples were above 10 AU/mL by SARS-CoV-2 IgG assay and below 1 COI by Roche’s kit. Among these samples, 12 cases were PCR-positive cases. In eight of these cases, the seroconversions were observed in SARS-CoV-2 IgG assay at earlier point than Roche’s kit, while, in other four cases, the antibody titers measured by Roche’s kit were below 1 COI although those at the former or the latter points were above 1 COI. These results suggest that false-negative cases in the CLIA assay for SARS-CoV-2 IgG might be fewer than Roche’s kit. According to the manufacturer’s instruction, the Roche’s kit adopts N protein as antigen to detect antibody and a principle of double antigen sandwich format. Therefore, the Roche’s kit theoretically detects all classes of antibodies against the antigen immobilized on beads [24]. Therefore, the small discrepancy in the concordance rate between Roche’s kit and SARS-CoV-2 IgM/IgG, especially IgM, may be derived from the difference in the principles of assays.

Regarding the antibody tests, in addition to issues surrounding accuracy, the matter of false-positive cases has also been a concern. As shown in Fig 3, the SARS-CoV-2 IgM/IgG levels were negative before hospitalization in three cases, and these antibody levels subsequently became positive after symptom onset in all cases, suggesting that this serological test can surely detect antibodies against SARS-CoV-2. In addition, we demonstrated that this measurement system could detect SARS-CoV-2 IgM/IgG without any false-positive results by evaluating 100 serum samples collected in 2017, when SARS-CoV-2 did not exist. These results confirm that this measurement system might be able to detect antibodies against SARS-CoV-2 alone, without cross-reactivity with other coronavirus strains that cause 15%-29% of all common colds [25, 26]. Chemiluminescence immunoassays are known to be affected by autoantibodies, such as rheumatoid factor, relatively often [27]. In the present study, we investigated whether five kinds of autoantibodies might interfere with the measurement system and found that no false-positive results were observed for the IgG assays, while four false-positive cases were observed for the IgM assays. We also found one PCR-negative case with a SARS-CoV-2 IgM titer over the cutoff value. Actually, when we investigated the reactivity of N protein and S protein to the sera separately, we found that the false-positive cases were caused by reactivity to the N protein (Table 4). The reason for this reactivity remains unclear at present but might be due to the cross-reactivity of the measurement system or, since the structure of the N protein of SARS-CoV-2 is similar to that of other coronavirus strains, antibodies to a structure similar to the N protein of SARS-CoV-2 might actually exist.

Finally, we investigated the concordance rate between PCR and this measurement system. As shown in Table 2, although all the PCR-negative subjects had negative results except for one subject with a high IgM level, the PCR-positive subjects did not necessarily have positive results for IgM or IgG. This discrepancy might be due to the fact that we used the serum sample that had been collected on the day nearest to the day on which the PCR sample had been collected. In several cases, only a few days had passed since the onset of symptoms, and IgG and IgM are reportedly not detectable during the early phase of COVID-19 [28]. Therefore, the time course for the appearance of IgG and IgM must be investigated for the application of antibody tests in clinical practice. It is difficult to compare the results of a molecular test and those of a serological assay because of the difference between the kinetics of viral RNA and those of antibodies. However, these results might help us understand the usefulness of the antibody testing in the diagnosis of COVID-19.

A characteristic of the present method is that both the N protein and the S protein are used as antigens. Reportedly, the sensitivity and specificity might depend on the types of antigens that are recognized by the antibodies, and the antibody to S protein is more sensitive than the antibody to N protein [29–31]. Since the measurement system in the present study uses both the S and N proteins, this system might provide a greater sensitivity and diagnostic ability than an antibody test using either the S protein or the N protein alone. In contrast, analyzing the reactivity to both proteins could increase the number of false-positive cases, as described above. Further studies on the clinical significances of antibodies to N protein and S protein might be necessary to conclude which is most appropriate: measuring the reactivity to both proteins or to each protein separately.

In conclusion, we have validated a measurement system for detecting IgM and IgG against SARS-CoV-2 using CLIA kits and have observed that this system had sufficient performance for its introduction into clinical laboratory testing. Moreover, the possibility of false-negative results, especially for IgG against SARS-CoV-2, was relatively low. In the future, this method might be helpful for clinical diagnosis, epidemiological investigations, and the development of vaccines.

Supporting information

S1 Data. This data set shows all the antibody data analyzed in the present study.

(XLSX)

Click here for additional data file. (411.9KB, xlsx)

Acknowledgments

We are thankful to the Murakami Foundation for the donation of the iFlash3000 to The University of Tokyo Hospital.

Abbreviations

ANOVA

analysis of variance

AU

arbitrary unit

CLIA

chemiluminescence immunoassay

CLSI

The Clinical & Laboratory Standards Institute

COI

cutoff index

COVID-19

coronavirus disease 2019

CV

coefficient variation

dsDNA

double-strand DNA

ECLIA

electrochemiluminescence immunoassay

FDA

the Food and Drug Administration

IgG

immunoglobulin G

IgM

immunoglobulin M

M2

mitochondrial M2

N protein

Nucleocapsid protein

P-ANCA

myeloperoxidase antineutrophil cytoplasmic antibody

RF

rheumatoid factor

RLU

relative light units

SARS-CoV-2

severe acute respiratory syndrome coronavirus 2

SD

standard division

S protein

Spike protein

SS-A

Sjögren’s syndrome A

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was conducted by the collaborative research project among The University of Tokyo, Shenzhen YHLO Biotech Co., Ltd, and Medical & Biological Laboratories Co. This work is also supported by the Research Grants in the Natural Sciences, The Mitsubishi Foundation awarded to M.K. F. X. and F. H. are employees of Shenzhen YHLO Biotech Co., Ltd and Y. K. and J. O. are employees of Medical & Biological Laboratories Co., Ltd.The funders provided support in the form of salaries for authors (F. X., F. H., Y. K., and J. O.), but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

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Decision Letter 0

Katerina Kourentzi

4 Sep 2020

PONE-D-20-21741

Validation of a new automated chemiluminescent anti-SARS-CoV-2 IgM and IgG antibody assay system detecting both N and S proteins in Japan

PLOS ONE

Dear Dr. Kurano,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

In addition to the issues raised by the reviewer I would like to note the following:

1) To formally evaluate and do a verification of the analytical and clinical performance of a quantitative test specific guidelines need to be followed: e.g. CLSI EP15-A3. The authors' conclusion that the performance of the system is sufficient is not substantiated by sufficient data.

2) How was the number of the samples chosen?

3) Basic assay background details are missing, e.g. chemiluminescence details (dye?), volumes used, calibrators, correlation between light units and AU antibody/ml.

4) Typically a new method needs to be compared to a gold standard- maybe a validated ELISA in this case? PCR is not an appropriate comparison for this assay.

5) How was serum collected and processed?

6) No formal estimation of LOB and LOD, LOQ  has been presented.

7) Linearity studies should be performed according to CLSI EP-06.

8) Figure 3- how were the "pre-infection" samples  in the 3 patients collected?

9) Line 115- typo "coLLected"

10) A quick search shows that the YHLO chemiluminescent anti-SARS-CoV-2 Ab assay is under FDA EUA and CE marked.

==============================

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Kind regards,

Katerina Kourentzi, PhD

Academic Editor

PLOS ONE

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In addition, please specify whether your IRB specifically approved your informed consent plan (opt-out on website).

3. Thank you for including your competing interests statement; "The present study is a collaborative research project among The University of Tokyo, Shenzhen YHLO Biotech Co., Ltd, and Medical & Biological Laboratories Co., Ltd. F. X. and F. H. are employees of Shenzhen YHLO Biotech Co., Ltd and Y. K. and J. O. are employees of Medical & Biological Laboratories Co., Ltd."

We note that one or more of the authors are employed by a commercial company: Shenzhen YHLO Biotech Co., Ltd, Medical & Biological Laboratories Co., Ltd.

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Reviewer #1: Yes

**********

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Reviewer #1: Yes

**********

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Reviewer #1: Yes

**********

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Reviewer #1: The study lacks originality. Even if the new should be linked to the application to a Japonase population , this is not enough. I suggest to make it as concise report or letter and submit to a different journal.

Some techinical details should be better explained in the text such as how they tested the antibodies against the single antigen (N or S) and why they used the non reactive serum to diluite the positive serum . The kit istruction don't say this and it's a strong action since they made linearity studies.

**********

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PLoS One. 2021 Mar 4;16(3):e0247711. doi: 10.1371/journal.pone.0247711.r002

Author response to Decision Letter 0

29 Oct 2020

Response to the Review Comments to the Author

Reviewer #1: The study lacks originality. Even if the new should be linked to the application to a Japonase population , this is not enough. I suggest to make it as concise report or letter and submit to a different journal.

Some techinical details should be better explained in the text such as how they tested the antibodies against the single antigen (N or S) and why they used the non reactive serum to diluite the positive serum . The kit istruction don't say this and it's a strong action since they made linearity studies.

We appreciate the critical point raised from Reviewer #1. We should admit that many articles have investigated the validity and usefulness of antibody tests, however since the clinical features of COVID-19 vary among countries, we believe it is an important task to investigate the validity and utility of antibody tests in Japan, where few researches have investigated those of antibody tests.

We apologize for the insufficient description on how to measure the antibody titers against the single antigen. We measured those antibody titers using magnetic beads coated with either antigen, respectively. The basic assay procedure is the same as the method of the original kit. We added this point to the Material and Method section in the revised manuscript (line 144 – 145).

We used non-reactive serum to dilute the positive serum, since a hook effect was observed in the measurement of SARS-CoV-2 IgG when we dilute the samples with saline. We added this result to the revised manuscript (line 233 – 236, Figure 2C).

Response to the Editor’s comments’

In addition to the issues raised by the reviewer I would like to note the following:

1) To formally evaluate and do a verification of the analytical and clinical performance of a quantitative test specific guidelines need to be followed: e.g. CLSI EP15-A3. The authors' conclusion that the performance of the system is sufficient is not substantiated by sufficient data.

We appreciate this suggestion by the Editor. We performed the verification of precision, using two kinds of samples each assay for 5 days, 5 replicates per run, according to the CLSI document EP15-A3.

Repeatability and within-laboratory precision of all samples were lower than 5% and the upper verification limits described in the original data published by the manufacture (Ref. 20). We added these results to the revised manuscript (line 197 – 207 and Table 2).

2) How was the number of the samples chosen?

We apologize for the insufficient description on how to choose the number of the samples. We enrolled the COVID-19 subjects and the suspicious subjects, which we can collect their residual serum samples between April 22 and June 22. We added this point in the revised manuscript (line 118 – 119)

3) Basic assay background details are missing, e.g. chemiluminescence details (dye?), volumes used, calibrators, correlation between light units and AU antibody/ml.

We apologize for the insufficient description on the assay information. These assays required 5 uL of the samples and the acridinium-labeled anti-human IgM or IgG conjugate antibody was used to detect them. The unites “AU/mL” are determined by comparing the RLU detected by iFlash optical system with the cutoff calculated from the SARS-CoV-2 IgM or IgG calibrators containing anti-SARS-CoV-2 IgM or IgG chimeric antibody. We added this information to the revised manuscript (line 136 – 142).

4) Typically a new method needs to be compared to a gold standard- maybe a validated ELISA in this case? PCR is not an appropriate comparison for this assay.

We appreciate this point raised from Editor. Unfortunately, because of the biosafety facility in our laboratory, we are unable to perform a manual-based ELISA to measure the antibody titers of COVID-19 patient. Instead of ELISA, we compared the chemiluminescent anti-SARS-CoV-2 antibody tests with the Roche’s COVID-19 antibody test, which received FDA emergency use authorization and was available in markets accepting the CD mark. According to the manufacturer’s instruction of the Roche’s test, the cutoff value for a positive COVID-19 antibody result was deemed as 1.0 cutoff index (COI). The positive and negative concordance rates between Roche’s test and SARS-CoV-2 IgM or IgG serological testing showed more than 85%, except for the positive concordance rate of SARS-CoV-2 IgM. We added these results to the Method section, the Result section, and the Discussion section in the revised manuscript (line 148 – 153, line 310 – 315, and line 360 – 371 and Table 4).

5) How was serum collected and processed?

We apologize for the lack of the description on how to collect serum. We collected the sera in the following steps. We received the patient’s whole blood in the collection tube coated with silica and thrombin for the clinical laboratory testing. Then, the serum was separated by centrifuging at 2,300 g for 5 minutes and carried out clinical routine testing. Subsequently, we collected and stored the residual samples at -20℃ until the antibody testing. We added these pointes in the revised manuscript (line112 – 115).

6) No formal estimation of LOB and LOD, LOQ has been presented.

We appreciate this comment from the Editor. As suggested by Editor, we added the formal estimation of LoB, LoD and LoQ, according to the guidelines in CLSI document EP17. The LoD for SARS-CoV-2 IgM was 0.74 AU/mL, determined by 130 measurements with 60 blank and 70 low level replicates, and the LoB was 0.63 AU/mL. The LoD for SARS-CoV-2 IgG was 0.53 AU/mL, determined by 120 measurements with 60 blank and 60 low level replicates, and the LoB was 0.47 AU/mL. We also investigated the LoQ. We measured 7 samples of low antibody levels for SARS-CoV-2 IgM and 10 samples for SARS-CoV-2 IgG. In both assays, CVs of the samples which were lower than the LoD showed no more than 10%. Therefore, the LoQ for SARS-CoV-2 IgM was determined as 0.74 AU/mL and that for SARS-CoV-2 IgG was 0.53 AU/mL. We added those results in the revised manuscript (line 175 – 182 and line 253 – 262).

7) Linearity studies should be performed according to CLSI EP-06.

We appreciate this comment from the Editor. As suggested by Editor, we performed the linearity studies, following the guidelines in CLSI document EP06. For IgM, the method was demonstrated to be linear between 1.50 and 15.92 AU/mL since any nonlinear coefficients were not significant in this range. And for IgG, the linear range was between 2.36 and 18.30 AU/mL since any nonlinear coefficients were not significant in this range. We added these results in the revised manuscript (line 225 – 229 and Figure 1E, F).

8) Figure 3- how were the "pre-infection" samples in the 3 patients collected?

We apologize for the insufficient information on how to collect the “pre-infection” samples. In our laboratory, the sera which completed clinical laboratory test were usually stored at -20℃ up to 3 weeks. We collected these samples since the sera which were collected and stored in our laboratory before the onset of COVID-19 symptom by chance. We added those points in the revised manuscript (line 115 – 118).

9) Line 115- typo "collected

"

We apologize for a misspelling. We have corrected this part. Thank you very much for kind suggestion (line 123).

10) A quick search shows that the YHLO chemiluminescent anti-SARS-CoV-2 Ab assay is under FDA EUA and CE marked.

We appreciate this kind information. Exactly, the YHLO chemiluminescent anti-SARS-Cov-2 Antibody assay acquired these authorizations. We believe that the evaluation in academic organizations is important to establish the clinical usefulness of these assays.

Response to the Journal Requirement

1. Please clarify whether all samples used in this study were stored biological samples, and whether samples were de-identified before researchers accessed them.

In addition, please specify whether your IRB specifically approved your informed consent plan (opt-out on website).

We have clarified these points on line 124 – 125 and line 127 – 128 in the revised manuscript.

2. Thank you for including your competing interests statement; "The present study is a collaborative research project among The University of Tokyo, Shenzhen YHLO Biotech Co., Ltd, and Medical & Biological Laboratories Co., Ltd. F. X. and F. H. are employees of Shenzhen YHLO Biotech Co., Ltd and Y. K. and J. O. are employees of Medical & Biological Laboratories Co., Ltd."

We note that one or more of the authors are employed by a commercial company: Shenzhen YHLO Biotech Co., Ltd, Medical & Biological Laboratories Co., Ltd.

Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

We have added the commercial affiliation in the section of funding sources (line 426 – 427) and included the requested statement (line 428 – 431).

We also revised the author contributions section (line 436 – 438) in more detail and update author roles in the online submission form.

2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

As requested, we revised the Competing Interests Statement (line 446 – 447) and added the requested statement (line 448 – 451).

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We apologized not to include the supporting information files for the data set. We submitted the file.

Attachment

Submitted filename: Response_yokoyama_final2.pdf

Click here for additional data file. (174.3KB, pdf)

Decision Letter 1

Katerina Kourentzi

8 Dec 2020

PONE-D-20-21741R1

Validation of a new automated chemiluminescent anti-SARS-CoV-2 IgM and IgG antibody assay system detecting both N and S proteins in Japan

PLOS ONE

Dear Dr. Kurano,

Thank you for submitting your revised manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Katerina Kourentzi, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The study of Yokoyama and colleagues is about a validation of a chemiluminescent assay for detection of antibodies IgM and IgG against SARS-CoV-2. Although there is a big importance in the validation of new methods to help in Covid-19 diagnosis, the study has some deficiencies regarding the study design.

1) To assess the applicability of the method using real patient samples, serum samples from 26 participants with confirmed PCR and 53 from suspect cases without laboratory confirmation were included. I believe that a larger and better characterized sample panel should have been used for this evaluation. Furthermore, authors should inform readers about the demographic and clinical profile of such participants, as well as for the negative controls (collected before the period and with autoimmune diseases) in a table.

2) Still regarding the positive participants samples, analysis of the sensitivity of the assay was made according to the PCR result and the authors only give the information that serum samples were collected a few days after PCR confirmation. What is the mean number of days of collection post symptoms onset? The first reviewer also mentioned the difficulty of compare a molecular and a serological assay because of the RNA and antibodies dynamics and suggested the use of a commercial ELISA method, which was incorporated to the analysis.

3) It is not clear for me why in the comparison of CLIA and the assay from Roche, much more positive serum samples were used. Is it possible to access PCR results from these individuals? Please include more characteristics of the samples and participants.

4) Authors explain that samples collected before the onset of the infection were collected by chance. Did you have performed any laboratorial detection assay to guarantee that these subjects did not have current or past SARS-CoV-2 infection at that time? The same question can be applied for negative controls collected on March 2020.

5) For the specificity analysis of the CLIA assay, is it possible to include samples from participants with confirmed coronavirus otherwise than SARS-COV-2 and also with other respiratory infections? The analysis of specificity with autoantibodies was very important.

6) The method validation is well described and after first revision, it was included a validation according to a protocol (EP15-A3). Authors should rethink if it is necessary both analysis or only the validated one.

7) Please include citation of Table 2A in line 196.

8) It is not clear for me how future researchers will interpret a negative result on CLIA as a possible situation of high titers of antibodies and false negativity. How can this work on the routine clinical practice?

9) In figure 3, authors showed the IgM and IgG dynamics before and after PCR confirmation. I’m not sure if data of only three participants are strong for this analysis. Furthermore, how do you explain the low titers of IgM in 2 of 3 of these participants?

10) The concordance of CLIA assay and Roche assay was not very good mainly for IgG detection. How do you interpret this divergence? So, I don’t agree with the sentence in the discussion lines 365 and 366.

11) Please include the new information of the study in the abstract section.

**********

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Reviewer #2: No

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PLoS One. 2021 Mar 4;16(3):e0247711. doi: 10.1371/journal.pone.0247711.r004

Author response to Decision Letter 1

15 Jan 2021

Point-by-Point Responses to the Reviewers’ Comments

For PONE-D-20-21741R1

#Correction

First of all, we apologize for inappropriate interpretation in the previous version that the samples of which the titers were equal to the cutoff value had been regard as positive for SARS-CoV-2 IgM/IgG assay and Roche’s kit. In this revised manuscript, we calculated these results again and as a result one sample was actually positive for Roche’s kit. We revised Table 3 and corrected the numbers of samples and concordance rates in line 308-312.

Response to the Review Comments to the Author

Reviewer #2: The study of Yokoyama and colleagues is about a validation of a chemiluminescent assay for detection of antibodies IgM and IgG against SARS-CoV-2. Although there is a big importance in the validation of new methods to help in Covid-19 diagnosis, the study has some deficiencies regarding the study design.

1) To assess the applicability of the method using real patient samples, serum samples from 26 participants with confirmed PCR and 53 from suspect cases without laboratory confirmation were included. I believe that a larger and better characterized sample panel should have been used for this evaluation. Furthermore, authors should inform readers about the demographic and clinical profile of such participants, as well as for the negative controls (collected before the period and with autoimmune diseases) in a table.

The samples were collected from subjects with negative PCR results at the same day as the PCR test was performed and there were no subjects suspected of autoimmune diseases. For the outpatients who had visited The University of Tokyo Hospital, we have chosen the subjects without autoimmune diseases. We added this information in line 127.

2) Still regarding the positive participants samples, analysis of the sensitivity of the assay was made according to the PCR result and the authors only give the information that serum samples were collected a few days after PCR confirmation. What is the mean number of days of collection post symptoms onset? The first reviewer also mentioned the difficulty of compare a molecular and a serological assay because of the RNA and antibodies dynamics and suggested the use of a commercial ELISA method, which was incorporated to the analysis.

The mean days (±S.D.) between the antibody test and the onset of the symptom or the PCR test were 11.3 (±6.70) or 5.67 (±5.67) days, respectively. We added this information in line 125-127.

Of course, we admit that the difficulty in comparing a molecular and a serological assay because of the RNA and antibodies dynamics. However, the aim of the present study is not only to validate our assays but also to investigate the usefulness in the diagnosis. Therefore, we compared the results of our assay with those of PCR tests. We added the difficulty in comparing both results in the revised manuscript (line 418-421).

Regarding the comparison with an ELISA, we compared out results with the results of FDA-approved assay from Roche, since manual based ELISAs have not been established to measure the antibodies against SARS-CoV-2 in Japan in addition to the difficulty in performing manual based ELISAs from the aspects of bio safety in our laboratory.

Judging from the results in the comparison with the Roche’s assay which measured antibodies without separating immunoglobulin subclasses, IgG test had a rather high concordance with the previous assay.

3) It is not clear for me why in the comparison of CLIA and the assay from Roche, much more positive serum samples were used. Is it possible to access PCR results from these individuals? Please include more characteristics of the samples and participants.

We apologize for the insufficient description about the characteristics of samples and participants we used in the comparison of CLIA and the assay from Roche. For this experiment (new Table 3B), we used all the serum samples which were collected from COVID-19-positive patient defined as the method section. In some cases, several serum samples collected from one patient were used.

To avoid this concern, we added the results of the concordance rates when we limited the samples only to the same samples as used in Table 2 (new Table 3A).

4) Authors explain that samples collected before the onset of the infection were collected by chance. Did you have performed any laboratorial detection assay to guarantee that these subjects did not have current or past SARS-CoV-2 infection at that time? The same question can be applied for negative controls collected on March 2020.

Two of those subjects had been confirmed SARS-CoV-2 PCR negative before the onset of COVID-19 symptoms. In one subject and control cases collected in March 2020, symptoms of cold were not described in medical records. In the revised manuscript, we added four cases of whom the samples collected before the onset of COVID-19 were available. One of the additional cases was confirmed PCR-negative before the onset of COVID-19 symptoms and regarding other cases, symptoms of cold were not described in medical records. We added this detail information on the subjects in line 119-122 in the revised manuscript.

5) For the specificity analysis of the CLIA assay, is it possible to include samples from participants with confirmed coronavirus otherwise than SARS-COV-2 and also with other respiratory infections? The analysis of specificity with autoantibodies was very important.

We agree that this is important point. At present, we did not obtain the serum of subjects infected with other coronaviruses. Instead, the manufacturer had assessed the cross-reactivity using serum samples of the subjects who had been confirmed with other respiratory infection (please see FYI Figures). These results suggested that these antibody tests did not have cross-reactivities against coronavirus other than SARS-CoV-2, such as OC43 and HKU1. These raw data, however, had not been permitted to be published by Shenzhen YHLO Biotech Co., Ltd. Therefore, we did not include these data in this paper.

6) The method validation is well described and after first revision, it was included a validation according to a protocol (EP15-A3). Authors should rethink if it is necessary both analysis or only the validated one.

We appreciate this kind suggestion. We will include the results of validation according to the CLSI document EP15-A3 and deleted the other results.

7) Please include citation of Table 2A in line 196.

We appreciate this kind suggestion. We added the citation in line 197 (new Table 1 in the revised manuscript).

8) It is not clear for me how future researchers will interpret a negative result on CLIA as a possible situation of high titers of antibodies and false negativity. How can this work on the routine clinical practice?

We appreciate the critical point raised from the Reviewer #2. Actually, the results on Figure 2C might propose the possibility that extremely high titers of IgG might result in false-negative result. However, to our experience, IgG titers of any participants who admitted to The University of Tokyo Hospital for COVID-19 (n = 83) had never become negative for SARS-CoV-2 IgG testing during the hospitalization once the seroconversion of IgG was observed. Therefore, we think that it would not be necessary to consider false negative results of IgG due to the extremely high titers and to dilute the serum samples of negative SARS-CoV-2 IgG results on the routine clinical practice. The simultaneous measurement of IgM would help the researchers to rule out the false negativity in IgG tests, since a hook effect was not observed in the SARS-CoV-2 IgM assay. We added this point in the Discussion section (line 360-365).

9) In figure 3, authors showed the IgM and IgG dynamics before and after PCR confirmation. I’m not sure if data of only three participants are strong for this analysis. Furthermore, how do you explain the low titers of IgM in 2 of 3 of these participants?

In the revising the manuscript, we enrolled four more participants of whom serum samples before and after PCR conformation were available. These additional results showed that SARS-CoV-2 IgM/IgG titers were changed from the titers below the cut-off level to those over the cut-off value at all 7 participants.

Regarding the low levels of IgM titers, the titers become over cutoff value in 4 of 7 of those participants. We rearranged the scales of SARS-CoV-2 IgM and IgG titers to present the results clearly (Figure 3). We revised the manuscript (line 262-264).

10) The concordance of CLIA assay and Roche assay was not very good mainly for IgG detection. How do you interpret this divergence? So, I don’t agree with the sentence in the discussion lines 365 and 366.

We apologize for the insufficient description about the concordance rate of CLIA assay and Roche’s kit. We added the detail of the samples of which the results were not concordant (line 374-383); three samples of which the antibody titers were below 10 AU/mL by SARS-CoV-2 IgG assay and above 1 COI by Roche’s kit were PCR-positive subjects, while we also observed seroconversion by SARS-CoV-2 IgG assay in the samples collected from the three subjects after 2 days. The antibody titers of 24 samples were above 10 AU/mL by SARS-CoV-2 IgG assay and below 1 COI by Roche’s kit. Among these samples, 12 cases were PCR-positive cases. In eight of these cases, the seroconversions were observed in SARS-CoV-2 IgG assay at earlier point than Roche’s kit, while, in other four cases, the antibody titers measured by Roche’s kit were below 1 COI although the antibody titers at the former or the latter points were above 1 COI.

Thinking these points, false-negative cases in the CLIA assay for SARS-CoV-2 IgG were fewer than Roche’s assay. We added this point in the revised manuscript (line 383-384).

Also, we deleted the description in line 365 and 366 in the previous manuscript according to the suggestion from the Reviewer #2.

11) Please include the new information of the study in the abstract section.

We appreciate this comment from the Reviewer #2. As suggested from Reviewer #2, we added the new information in the abstract section (line 62-64).

Attachment

Submitted filename: Response-yokoyamaR2.pdf

Click here for additional data file. (630.5KB, pdf)

Decision Letter 2

Katerina Kourentzi

12 Feb 2021

Validation of a new automated chemiluminescent anti-SARS-CoV-2 IgM and IgG antibody assay system detecting both N and S proteins in Japan

PONE-D-20-21741R2

Dear Dr. Kurano,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Katerina Kourentzi, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

**********

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Acceptance letter

Katerina Kourentzi

23 Feb 2021

PONE-D-20-21741R2

Validation of a new automated chemiluminescent anti-SARS-CoV-2 IgM and IgG antibody assay system detecting both N and S proteins in Japan

Dear Dr. Kurano:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr. Katerina Kourentzi

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Data. This data set shows all the antibody data analyzed in the present study.

    (XLSX)

    Click here for additional data file. (411.9KB, xlsx)
    Attachment

    Submitted filename: Response_yokoyama_final2.pdf

    Click here for additional data file. (174.3KB, pdf)
    Attachment

    Submitted filename: Response-yokoyamaR2.pdf

    Click here for additional data file. (630.5KB, pdf)

    Data Availability Statement

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