Serological tests for detection of anti–severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Abs in blood are expected to identify individuals who have acquired immunity against SARS-CoV-2 and indication of seroprevalence of SARS-CoV-2 infection. Many serological tests have been developed to detect Abs against SARS-CoV-2. However, these tests have considerable variations in their specificity and sensitivity, and whether they can predict levels of neutralizing activity is yet to be determined. This study aimed to investigate the kinetics and neutralizing activity of various Ag-specific Ab isotypes against SARS-CoV-2 in serum of coronavirus disease 2019 (COVID-19) patients confirmed via PCR test. We developed IgG, IgM, and IgA measurement assays for each Ag, including receptor-binding domain (RBD) of spike (S) protein, S1 domain, full-length S protein, S trimer, and nucleocapsid (N) domain, based on ELISA. The assays of the S protein for all isotypes showed high specificity, whereas the assays for all isotypes against N protein showed lower specificity. The sensitivity of all Ag-specific Ab isotypes depended on the timing of the serum collection and all of them, except for IgM against N protein, reached more than 90% at 15–21 d postsymptom onset. The best correlation with virus-neutralizing activity was found for IgG against RBD, and levels of IgG against RBD in sera from four patients with severe COVID-19 increased concordantly with neutralizing activity. Our results provide valuable information regarding the selection of serological test for seroprevalence and vaccine evaluation studies.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has instigated a widespread concern globally (1). As of November 1, 2020, more than 46 million confirmed cases and 1 million deaths have been recorded worldwide. Currently, the gold standard for the diagnosis of COVID-19 is real time RT-PCR assay, which detects SARS-CoV-2 RNA in respiratory tract specimens. In the meantime, serological tests to detect anti–SARS-CoV-2 Abs in the blood have also been investigated (2, 3). Although the immune response and Ab kinetics against SARS-CoV-2 are not well understood, serological tests are expected to be useful for various purposes (4, 5). For instance, convalescent serum/plasma therapy has been initiated for patients with severe COVID-19 in some countries (6, 7). Serological tests that quantify neutralizing Abs should effectively identify donors with high Ab titers. Serological tests for neutralizing Abs may also be used to determine the efficacy of vaccines currently being developed (810). In addition, serological tests provide insight into an individual’s immune status as well as important epidemiological information on the spread of the infection in a specific region (2).

To measure an Ab against SARS-CoV-2, it is important to measure an Ag-specific Ab suitable for its application. A typical coronavirus (CoV) contains four major structural proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N) (11, 12). Previous studies have reported that highly immunogenic Ags of SARS-CoV and Middle East respiratory syndrome CoV (MERS-CoV) are S and N proteins (1316). Therefore, most of the serological assays developed to detect anti–SARS-CoV-2 Ab also target these proteins. The S protein consists of two functional subunits (S1 and S2), which form a homotrimer. S1 contains a receptor-binding domain (RBD) and is required for binding to host cells. Therefore, detection of S1 (RBD)-specific Ab is considered as an index for predicting neutralizing activity that protects host cells from virus infection (17, 18). In contrast, N protein functions to package the capsid-formed genome into virions and acts as a viral RNA-silencing suppressor required for viral replication (12). The N protein of CoV is found to be overexpressed in large quantities during infection and is highly immunogenic. Therefore, Abs against the N protein are promising indicators for detecting SARS-CoV-2 infection, especially once SARS-CoV-2 vaccines are widely deployed, all of which currently uses whole or partial S protein as the Ag.

To date, based on S and N proteins, many serological diagnostic methods have been developed to detect Abs against SARS-CoV-2. However, detailed analysis of Ab isotypes against each Ag, including the S trimer, has not been conducted. Therefore, this study aimed to investigate the kinetics and neutralizing activity of various Ag-specific Abs against SARS-CoV-2 in the serum of PCR-confirmed COVID-19 patients. To assess the various Ag-specific Abs, we developed the IgG, IgM, and IgA measurement assays for each Ag, including RBD, S1, S full length (S full), S trimer, and N based on ELISA. Using this method, we systematically analyzed the Abs in the serum of COVID-19 patients. We also examined the correlation between each Ab and the neutralizing activity to clarify which Ab best predicted neutralizing activity. This study advances the understanding of immune response and Ab kinetics against SARS-CoV-2 and informs several applications such as identifying donors with high Ab titers for convalescent serum/plasma therapy and measuring the immunogenicity of vaccines being developed.

This study was reviewed and approved by the Ethics Committee for Clinical Research of the Center for Research Promotion and Support in Fujita Health University (authorization number HM19-493 and HM17-341).

We used a series of residual serum samples from 41 COVID-19 patients who were admitted to Fujita Health University Hospital from February 28, 2020, to May 21, 2020. The demographic and clinical characteristics of the patients are presented in Table I and Supplemental Table I. All patients were confirmed as COVID-19 cases by real time PCR assay of nasopharyngeal swab specimens at the time of or prior to admission. The date of onset was determined as the day when the patients started experiencing COVID-19 symptoms. Severity classifications were made according to the Guidelines of the Treatment and Management of Patients with COVID-19 published by the Infectious Diseases Society of America. patients with severe COVID-19 were defined as those with SpO2 ≤ 94% on room air, or those who require supplemental oxygen. The other patients were defined as having nonsevere COVID-19.

Table I.

Clinical characteristics of COVID-19 patients

Total (n = 41)Severe (n = 6)Nonsevere (n = 35)
No.%No.%No.%
Age, median (minimum–maximum) 41 y (16–93 y) 54 y (31–66 y) 53 y (16–93 y) 
Sex       
 Male 22 53.7 50.0 19 54.3 
 Female 19 46.3 50.0 16 45.7 
Symptoms       
 Fever 33 80.5 100.0 27 77.1 
 Cough 12 29.3 50.0 25.7 
 Dyspnea 9.8 66.7 0.0 
 Diarrhea 2.4 16.7 0.0 
 Dysgeusia 4.9 0.0 5.7 
 Dysosmia 9.8 0.0 11.4 
 Sore throat 4.9 0.0 5.7 
 Asymptomatic 7.3 0.0 8.6 
Total (n = 41)Severe (n = 6)Nonsevere (n = 35)
No.%No.%No.%
Age, median (minimum–maximum) 41 y (16–93 y) 54 y (31–66 y) 53 y (16–93 y) 
Sex       
 Male 22 53.7 50.0 19 54.3 
 Female 19 46.3 50.0 16 45.7 
Symptoms       
 Fever 33 80.5 100.0 27 77.1 
 Cough 12 29.3 50.0 25.7 
 Dyspnea 9.8 66.7 0.0 
 Diarrhea 2.4 16.7 0.0 
 Dysgeusia 4.9 0.0 5.7 
 Dysosmia 9.8 0.0 11.4 
 Sore throat 4.9 0.0 5.7 
 Asymptomatic 7.3 0.0 8.6 

One hundred serum samples obtained from 100 healthy human volunteers (mean age, 47; males, 58 and females, 42), collected before the COVID-19 pandemic (July 2012), were used as negative controls to evaluate the specificity and cut-off values for each assay. All serum samples (aliquoted and stored at –80°C) were thawed and evaluated at the same time for the analyses.

Recombinant SARS-CoV-2 RBD, S1, S trimer, and N proteins, expressed in HEK293 cells were purchased from Acro Biosystems. These recombinant proteins carry a polyhistidine tag at the C terminus, which are used to purify proteins. Recombinant S trimer contains alanine substitutions (R683A and R685A), which are introduced to stabilize the trimeric prefusion state of the S protein and abolish the furin cleavage site, respectively. Recombinant S full protein (full sequence of S protein), expressed and purified from HEK293 cells, was purchased from CerTest BIOTEC (Zaragoza, Spain). The 96-well plates (Thermo Fisher Scientific) were coated per well with 250 ng of individual recombinant proteins, covered, and incubated overnight at 4°C. The plates were washed with PBS containing 0.1% Tween 20 (PBST) (FUJIFILM Wako Pure Chemical, Osaka, Japan), blocked with PBST containing 1% casein protein stored overnight at 4°C. After blocking, plates were vacuum dried.

Serum samples were heated at 56°C for 30 min before use to inactivate any potential residual virus in the sera. Serum samples were diluted by 1:201 for IgG and IgA assays or 1:2010 for IgM assay in sample buffer containing 2% BSA. Afterwards, 50 µl of the diluted serum samples were added per well and incubated at room temperature (RT) for 60 min. The plates were washed three times with 300 µl PBST and 50 µl of peroxidase-labeled anti-human IgG (FUJIFILM Wako Pure Chemical, Osaka, Japan), IgM, or IgA Ab (Midrand Bioproducts) was added per well and incubated at RT for 60 min. After incubation, the plates were washed five times with 300 µl PBST and 100 µl of substrate solution (TMB/H2O2) (FUJIFILM Wako Pure Chemical, Osaka, Japan) was added per well and incubated at RT for 10 min. Subsequently, the reaction was stopped by addition of 100 µl of 1 M HCl. The absorbance was measured at 450 nm using a 620-nm reference filter in a microplate reader (Thermo Fisher Scientific).

Serum samples from 10 COVID-19 patients who had strong IgG and IgA positivity and five patients who had strong IgM positivity were pooled and used to prepare positive reference standards for IgG, IgA, and IgM. The standard pooled serum was diluted (1:201 for IgG and IgA and 1:2010 for IgM) with sample buffer containing 2% BSA. The standard sera were diluted to 1:4, 1:6, 1:8, and 1:16 with sample buffer containing 2% BSA. These serially diluted standard sera were aliquoted and maintained at –80°C until use. The diluted standard sera were applied to an ELISA plate and ELISA was performed in the same way as for the test samples to make a standard curve. The OD450 of the standard sera of 0.015 was converted to 1 U/ml and these values were fitted to a line graph using linear regression analysis. The Ab units in test serum samples were then calculated from their OD450 values using the parameters estimated from the standard curve.

Serum samples were heat-inactivated at 56°C for 30 min and then serially diluted with DMEM (Fujifilm Wako Pure Chemical, Osaka, Japan) supplemented with 2% FBS (Biowest), 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, USA). The mixture of diluted sera and 100 tissue culture ID50 SARS-CoV-2 JPN/TY/WK-521 strain were incubated at 37°C for 1 h, then placed on VeroE6/TMRRSS2 cells (JCRB1819; Japanese Collection Research Bioresources Cell Bank) and cultured at 37°C with 5% CO2 (19). On day 5, plates were fixed with 20% formalin (Fujifilm Wako Pure Chemical, Osaka, Japan) and stained with crystal violet solution (Sigma-Aldrich) for evaluating the cytopathic effect. The index of the highest sera dilution factor with cytopathic effect inhibition was defined as the microneutralization test titer (MNT).

Statistical analysis was performed using GraphPad Prism version 8.0.0 for Windows (GraphPad Software). Sensitivity, specificity, and receiver operating characteristic (ROC) were calculated based on the RT-PCR results. Correlations analysis between Ab titer and neutralization test was performed using Spearman correlation coefficient. The statistical difference between patients with nonsevere and severe COVID-19 was determined using two-tailed Mann–Whitney U test and p < 0.05 was considered statistically significant.

We evaluated the ELISA designed to detect SARS-CoV-2 Ag-specific IgG, IgM, and IgA against RBD, S1, S full, S trimer, and N protein. To quantify the Ab responses to each Ag, we tested 169 serums from 41 SARS-CoV-2–infected patients and 100 negative control serums from healthy donor collected before SARS-CoV-2 pandemic. Fig. 1 shows the kinetics of the Ag-specific Ab isotype responses in each of the COVID-19 patients (6 severe and 35 nonsevere) in the days after the onset of symptoms. Longitudinal analysis showed similar kinetics among S proteins in most of the patients, but the isotypes against N proteins showed slightly different patterns among patients.

FIGURE 1.

Kinetics of Ag-specific Ab isotype response in each COVID-19 patient in the days after the onset of symptoms. The serum samples of 41 COVID-19 patients (6 severe and 35 nonsevere) from whom we had obtained sequential serum samples were analyzed.

FIGURE 1.

Kinetics of Ag-specific Ab isotype response in each COVID-19 patient in the days after the onset of symptoms. The serum samples of 41 COVID-19 patients (6 severe and 35 nonsevere) from whom we had obtained sequential serum samples were analyzed.

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Next, we categorized serums from COVID-19 patients according to the timing of their collection relative to symptom onset. All Ag-specific Abs and their isotypes started to increase as early as 0–7 d after the onset of symptoms compared with the negative control samples, which showed no marked response to any of the Ags (Fig. 2). The IgG and IgM levels against all Ags continued to increase over time, whereas IgA levels peaked by 15–21 d and declined thereafter.

FIGURE 2.

Ab isotype responses against each SARS-CoV-2 Ag in COVID19 patients. The serum samples from 41 COVID-19 patients (days 0–7 [n = 37], days 8–14 [n = 54], days 15–21 [n = 49] and days 22–47 [n = 29] from symptom onset) and control samples (n = 100) were tested for IgG, IgM, and IgA Abs against SARS-CoV-2 RBD, S1, S full, S trimer, and N Ags using ELISAs. Error bars represent SEMs.

FIGURE 2.

Ab isotype responses against each SARS-CoV-2 Ag in COVID19 patients. The serum samples from 41 COVID-19 patients (days 0–7 [n = 37], days 8–14 [n = 54], days 15–21 [n = 49] and days 22–47 [n = 29] from symptom onset) and control samples (n = 100) were tested for IgG, IgM, and IgA Abs against SARS-CoV-2 RBD, S1, S full, S trimer, and N Ags using ELISAs. Error bars represent SEMs.

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To evaluate the diagnostic performance and determine the cut-off values of the developed ELISAs, ROC analysis was performed using the same samples. The ROC analysis was conducted and the area under curve (AUC) was calculated using GraphPad Prism software version 8.0.0. The ROC analysis showed that the diagnostic performance differed according to the Ags and their isotypes (Fig. 3). The best results were obtained with S full-IgG (AUC = 0.943). Conversely, N-IgM showed the lowest diagnostic performance (AUC = 0.797). The analysis also showed that AUC of all Ag-specific Abs and their isotypes increased over time after the onset of symptoms (Fig. 3).

FIGURE 3.

ROCs analysis of Ab isotypes for each SARS-CoV-2 Ag over several time periods after symptom onset. COVID-19 negative (n = 100) and positive (n = 169) serum samples from 41 patients (days 0–7 [n = 37], days 8–14 [n = 54], days 15–21 [n = 49], and days 22–47 [n = 29] after symptom onset) were analyzed.

FIGURE 3.

ROCs analysis of Ab isotypes for each SARS-CoV-2 Ag over several time periods after symptom onset. COVID-19 negative (n = 100) and positive (n = 169) serum samples from 41 patients (days 0–7 [n = 37], days 8–14 [n = 54], days 15–21 [n = 49], and days 22–47 [n = 29] after symptom onset) were analyzed.

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To evaluate the specificity and sensitivity of the developed ELISAs, the optimal cut-off values for each Ag-specific Ab isotype were determined by the Youden index using the ROC analysis of all samples (Table II). The specificity of the S protein (RBD, S1, S full, and S Trimer) assays for all isotypes showed comparable results, with high specificity ranging from 94.0–99.0%. In contrast, the assays against N protein for all isotypes showed lower specificities of 79.0%, 88.0%, and 82.0% for N-IgG, N-IgM, and N-IgA, respectively. The sensitivity was dependent on the timing of the serum collection. During 0–7 d after symptom onset, the sensitivity of Ag-specific IgG, IgM and IgA were considerably different and ranged between 23.7–57.9%, 13.2–44.7%, and 31.6–50.0%, respectively. Thereafter, the sensitivity of Ag-specific IgG, IgM, and IgA increased to more than 90% at 15–21 d after symptom onset, except for N-IgM (85.7%). The sensitivity of all Ag-specific Ab isotypes increased to maximal levels at 22–47 d after symptom onset and ranged between 93.1–96.6%, except for N-IgM (79.3%).

Table II.

Specificity and sensitivity of Ab isotypes for each SARS-CoV-2 Ag based on time periods after symptom onset

Cutoff value (U/ml)RBDS1S FullS TrimerN
IgG Abs      
 Cut-off value (U/ml) 0.86 1.10 0.70 1.80 0.70 
 Specificity (negative control, n = 100) 98.0% 97.0% 93.0% 96.0% 79.0% 
 Sensitivity range (d)      
  0–7 d (n = 37) 39.5% 23.7% 47.4% 31.6% 57.9% 
  8–14 d (n = 54) 79.6% 72.2% 83.3% 79.6% 85.2% 
  15–21 d (n = 49) 93.9% 93.9% 95.9% 95.9% 95.9% 
  22–47 d (n = 29) 96.6% 96.6% 96.6% 96.6% 96.6% 
IgM Abs 
 Cut-off value (U/ml) 1.70 2.07 1.10 3.12 5.65 
 Specificity (negative control, n = 100) 94.0% 99.0% 94.0% 97.0% 88.0% 
 Sensitivity range (d)      
  0–7 d (n = 37) 36.8% 26.3% 44.7% 13.2% 21.1% 
  8–14 d (n = 54) 87.0% 85.2% 88.9% 68.5% 64.8% 
  15–21 d (n = 49) 93.9% 93.9% 95.9% 91.8% 85.7% 
  22–47 d (n = 29) 96.6% 96.6% 96.6% 93.1% 79.3% 
IgA Abs 
 Cut-off value (U/ml) 1.85 1.89 4.37 3.40 3.00 
 Specificity (negative control, n = 100) 97.0% 94.0% 99.0% 97.0% 82.0% 
 Sensitivity range (d)      
  0–7 d (n = 37) 36.8% 31.6% 34.2% 31.6% 50.0% 
  8–14 d (n = 54) 87.0% 87.0% 83.3% 83.3% 79.6% 
  15–21 d (n = 49) 95.9% 95.9% 93.9% 95.9% 93.9% 
  22–47 d (n = 29) 96.6% 96.6% 96.6% 96.6% 96.6% 
Cutoff value (U/ml)RBDS1S FullS TrimerN
IgG Abs      
 Cut-off value (U/ml) 0.86 1.10 0.70 1.80 0.70 
 Specificity (negative control, n = 100) 98.0% 97.0% 93.0% 96.0% 79.0% 
 Sensitivity range (d)      
  0–7 d (n = 37) 39.5% 23.7% 47.4% 31.6% 57.9% 
  8–14 d (n = 54) 79.6% 72.2% 83.3% 79.6% 85.2% 
  15–21 d (n = 49) 93.9% 93.9% 95.9% 95.9% 95.9% 
  22–47 d (n = 29) 96.6% 96.6% 96.6% 96.6% 96.6% 
IgM Abs 
 Cut-off value (U/ml) 1.70 2.07 1.10 3.12 5.65 
 Specificity (negative control, n = 100) 94.0% 99.0% 94.0% 97.0% 88.0% 
 Sensitivity range (d)      
  0–7 d (n = 37) 36.8% 26.3% 44.7% 13.2% 21.1% 
  8–14 d (n = 54) 87.0% 85.2% 88.9% 68.5% 64.8% 
  15–21 d (n = 49) 93.9% 93.9% 95.9% 91.8% 85.7% 
  22–47 d (n = 29) 96.6% 96.6% 96.6% 93.1% 79.3% 
IgA Abs 
 Cut-off value (U/ml) 1.85 1.89 4.37 3.40 3.00 
 Specificity (negative control, n = 100) 97.0% 94.0% 99.0% 97.0% 82.0% 
 Sensitivity range (d)      
  0–7 d (n = 37) 36.8% 31.6% 34.2% 31.6% 50.0% 
  8–14 d (n = 54) 87.0% 87.0% 83.3% 83.3% 79.6% 
  15–21 d (n = 49) 95.9% 95.9% 93.9% 95.9% 93.9% 
  22–47 d (n = 29) 96.6% 96.6% 96.6% 96.6% 96.6% 

Next, we examined the relationship between Ag-specific Ab isotype levels and virus-neutralizing activity in sera collected from the COVID-19 patients. We performed neutralization assays in a subset of samples including 34 sera from 19 COVID-19 patients. Because we used residual serum samples after completion of routine laboratory testing, a subset of samples that had enough volume to measure neutralizing activity was used for neutralization assay. Fig. 4A shows the correlation between each of the Ag-specific Ab isotypes and the neutralizing activity (MNT). All Ag-specific isotypes, except for N-IgM (Spearman r = 0.150, p = 0.397) and N-IgA (Spearman r = 0.172, p = 0.332), showed significant positive correlation between Ab levels and neutralizing activity (Fig. 4A). The best correlation was found with RBD-IgG (Spearman r = 0.751, p < 0.0001). Fig. 4B presents the Spearman correlation coefficient (r) of each Ag-specific Ab isotypes. Overall, IgG showed good correlation with neutralizing activity in all the Ags. Conversely, IgA showed a relatively lower correlation with neutralizing activity and N-IgA in particular showed no significant correlation.

FIGURE 4.

Correlation between Ag-specific Ab isotypes and neutralizing activity. (A) Correlation analysis between Ab titer for each SARS-CoV-2 Ag and MNT was calculated using Spearman correlation coefficient. Serological and neutralization assays were performed using 34 serums from 19 patients. (B) Graph represents the Spearman coefficient (r) in (A). #p > 0.1.

FIGURE 4.

Correlation between Ag-specific Ab isotypes and neutralizing activity. (A) Correlation analysis between Ab titer for each SARS-CoV-2 Ag and MNT was calculated using Spearman correlation coefficient. Serological and neutralization assays were performed using 34 serums from 19 patients. (B) Graph represents the Spearman coefficient (r) in (A). #p > 0.1.

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We categorized the COVID-19 patients into two severity groups (severe and nonsevere) based on established clinical classifications. The average levels of RBD-IgG in the serum samples, which was the best indicator of neutralizing activity according to Fig. 4, are shown in Fig. 5. Serum RBD-IgG levels in patients with severe COVID-19 were significantly higher than nonsevere cases as early as 8–14 d after symptom onset, whereas patients with severe COVID-19 produced high levels of RBD-IgG over time.

FIGURE 5.

Relationship between IgG Ab titer against RBD and disease severity. RBD-IgG were classified into two severity groups: severe and nonsevere. Red is severe (6 patients), and blue is nonsevere (35 patients). Statistical analysis was done by two-tailed Mann–Whitney test. **p < 0.01, ***p < 0.001.

FIGURE 5.

Relationship between IgG Ab titer against RBD and disease severity. RBD-IgG were classified into two severity groups: severe and nonsevere. Red is severe (6 patients), and blue is nonsevere (35 patients). Statistical analysis was done by two-tailed Mann–Whitney test. **p < 0.01, ***p < 0.001.

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We evaluated the relationship between RBD-IgG responses and virus-neutralizing activity in serum samples obtained from four patients with severe COVID-19 at several time points after symptom onset. They all recovered from COVID-19 and duration of the COVID-19-associated symptoms was 21, 23, 22, and 37 for patients 5, 14, 31, and 34, respectively. As shown in Fig. 6, serum RBD-IgG in all four patients increased over time, in concordance with virus-neutralizing activity.

FIGURE 6.

Kinetics of RBD-IgG Ab titers and neutralizing activity in patients with severe COVID-19. Relationship between RBD-IgG titers and MNTs in serum samples from four patients with severe COVID-19 over several time periods after symptom onset.

FIGURE 6.

Kinetics of RBD-IgG Ab titers and neutralizing activity in patients with severe COVID-19. Relationship between RBD-IgG titers and MNTs in serum samples from four patients with severe COVID-19 over several time periods after symptom onset.

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In this study, we comprehensively analyzed Ag-specific anti–SARS-CoV-2 Ab isotypes, including IgG, IgM, and IgA in COVID-19 patients and identified differential Ab responses and their relationship with neutralizing activity among the isotypes. To date, this is the first study, to our knowledge, that systematically analyzed reactivity of 15 different Ag-specific Ab isotypes in the serum samples of COVID-19 patients. The insights into the reactivity of each Ab isotype against various Ags generated in this study should further improve our understanding of the humoral immune responses against SARS-CoV-2.

We investigated the kinetics of RBD-, S1-, S full–, S trimer–, and N protein–specific IgG, IgM, and IgA responses in 169 serum samples obtained from 41 COVID-19 patients. Our study showed that Ag-specific Ab isotypes in the sera from several patients increased within 0–7 d after the onset of symptoms (Fig. 2 and Table II). The average of all Ag-specific IgA levels reached their peaks in 15–21 d after the onset of symptoms, whereas all Ag-specific IgG and IgM levels significantly increased in 8–14 d after the onset and continued to increase thereafter. These increases in IgG and IgM are consistent with some recent reports (2025). Although there are few available reports on the responses of IgA in COVID-19 patients, one report showed that RBD-specific IgA reached the peak during 16–20 d after the onset of symptoms, which is consistent with our findings (22). These results indicate that, although there are some differences among the isotypes, all Ag-specific Ab isotypes can be detected in the serum of most patients ?2 wk after the onset of symptoms.

However, our further analysis revealed that there are considerable differences among the Abs in terms of the specificity and sensitivity in predicting disease. When the specificity of each Ag-specific Ab isotypes was investigated using a cut-off value determined by the ROC analysis, specificity of the S protein assay for all isotypes showed high specificities ranging between 94.0–99.0%, whereas the N protein assay for all isotypes showed lower specificities of 79.0%, 88.0%, and 82.0% for N-IgG, N-IgM, and N-IgA, respectively. The lower specificity of N protein may be due to potential cross-reactivity with Abs to commonly circulating CoVs in the negative control samples. Although we have not addressed the potential cross-reactivity of each Ag with serum from humans infected with other CoVs, several studies have shown that N protein–based serological assays were more often associated with cross-reactivity than S protein–based assays (26). Chia et al. reported that S1 or RBD showed better specificity than N-based serological assay and that there was significant cross-reactivity when N protein was used as the Ag for their assay (27). Furthermore, Algaissi et al. reported that the specificity of N-based IgG and IgM assays showed lower sensitivity than S1-based assays (23). In addition, a recent study showed that Abs against the N protein declined more rapidly than those against RBD (28). Our findings support the use of S protein as the Ag for the detection of SARS-CoV-2–specific Abs.

We also determined which Ag-specific Ab isotype assays best represented the virus-neutralizing activity (Fig. 4A, 4B). Although most of the Ab isotypes showed good correlation with neutralizing activity, RBD-IgG assay showed the best correlation. These results are in line with the findings of other reports, which showed correlation of RBD-IgG levels with neutralizing activity (29, 30). Neutralization assays are difficult to perform as routine tests as they require viral cultures and must be performed in laboratories with higher biosafety levels. Measurement of RBD-IgG levels in the serum can be a reliable and convenient tool for assessing the immunological response of COVID-19 patients. We also suggest that measuring RBD-IgG levels in the serum may be a useful tool to identify donors for convalescent serum/plasma therapy and quantify the immunogenicity of vaccines, which are currently being developed (10, 31, 32).

Because RBD-IgG levels in the serum correlated with virus-neutralizing activity, we investigated the relationship between severity of COVID-19 and serum RBD-IgG levels. As shown in Fig. 5, RBD-IgG levels in the serum samples collected from patients with severe COVID-19 within 8–14 d after symptom onset were significantly higher than those of nonsevere patients; however, the patients with severe COVID-19 showed much higher levels of RBD-IgG thereafter. Consistent with previous reports, our results indicate that measurement of RBD-IgG levels correlate with disease severity of COVID-19 (22, 33). Furthermore, the serum RBD-IgG levels in patients with severe COVID-19 who recovered increased along with neutralizing activity. These results suggest that measurement of RBD-IgG levels in convalescent patients can be used to identify appropriate donors with high neutralizing activity for convalescent serum/plasma therapy (7, 10).

It has been reported that the RBD of SARS-CoV can elicit highly potent neutralizing Ab responses in immunized animals (34). Similar to SARS-CoV, SARS-CoV-2 entry into host cells is mediated by the RBD, which binds to angiotensin-converting enzyme 2 (3538); therefore, RBD is considered as the main target of neutralizing Abs and has been a focus of therapeutic and vaccine design efforts (10, 39, 40). Indeed, a recent study reported that SARS-CoV-2’s RBD is immunodominant and RBD-directed Abs accounted for more than 90% of the SARS-CoV-2–neutralizing humoral response (41). These reports support our suggestions that measurement of RBD-IgG levels may be a useful tool for identifying donors for convalescent serum/plasma therapy and quantifying the immunogenicity of vaccines that are currently being developed.

In some countries, convalescent serum/plasma therapy is currently being administered to patients with severe COVID-19 in some countries. For example, by late 2020, over 250,000 patients had been transfused with convalescent plasma in the US alone (42). The optimal selection of donors to ensure high titers of convalescent plasma is important. In fact, the US Food and Drug Administration has recommended the use of the Ortho Vitros SARS-CoV-2 IgG test (which uses S1 as a capture Ag) under emergency use authorization as the standard to qualify units of convalescent plasma. A recent study from Girardin et al. (43) reported that the neutralizing capacity of convalescent plasma can be determined by the Ortho Vitros SARS-CoV-2 IgG test. In addition to this test, RBD-IgG ELISA is also applicable to this use, and may even be better for the selection of convalescent plasma, because RBD-IgG showed better correlation with neutralizing activity than S1-IgG (Fig. 4A, 4B). Furthermore, ELISA can be used in large-scale screening and to exclude low titer donors.

Recently, several ELISAs have been developed to detect anti–SARS-CoV-2 Abs and have been used in clinical studies under the US Food and Drug Administration’s emergency use authorization. Most of them use the S protein (S full, S1, or RBD) as the capture Ag, but some use N protein. Our results indicated that the measurement of anti–SARS-CoV-2 Abs against the S protein, especially RBD-IgG, best correlate with virus-neutralizing activity and disease severity and, therefore, may be the optimal assay to assess the immunological response of COVID-19 patients. Meanwhile, it has been reported that the N protein of CoV is found to be overexpressed in large quantities during infection and is highly immunogenic (44). Therefore, Abs against the N protein may be better indicators for detecting SARS-CoV-2 infection than Abs against the S protein. In addition, the use of partial S protein may result in SARS-CoV-2 infection being missed because high levels of Abs can be generated by proteins other than to S1 or RBD (45). Therefore, we suggest that the selection of capture Ags should be carefully considered and that capture Ags should be selected appropriately depending on the situation.

There are several limitations in this study. Importantly, there were only four patients with severe COVID-19 from whom residual serum samples were available to investigate the association between serum RBD-IgG levels and neutralizing activity. Because these four patients with severe COVID-19 all recovered from COVID-19, we could not investigate differences in Ab responses and neutralizing activity between patients who recovered from COVID-19 and those who did not. Another limitation is that the heat inactivation of the serum samples may have interfered with the results. The serum samples in our study were heated at 56°C for 30 min before use to inactivate any potential residual virus; however, a recent study that used immunochromatographic assays showed that heat inactivation of serum at 56°C for 30 min interferes with the immunoreactivity of Abs to SARS-CoV-2; therefore, the possibility of false-negative results should be considered if the sample was preinactivated by heating (46). However, another report, which used ELISAs, showed that heat inactivation of serum at 56°C for 60 min had no negative impact on assay performance (20). The discrepancy between these findings may be due to differences in detection sensitivity between the immunochromatographic and ELISAs, but the effect of heat inactivation on the immunoreactivity of Abs to SARS-CoV-2 should be clarified using a larger number of samples in the future.

In summary, our results indicate that the anti–SARS-CoV-2 Ab response in COVID-19 patients varies among the Ag-specific Ab isotypes. Diagnostic performance of ELISAs for detection of anti–SARS-CoV-2 Abs also varies among Ag-specific Ab isotypes. Among them, serum RBD-IgG levels best correlate with virus-neutralizing activity and disease severity, thus may be the optimal assay to track COVID-19 seroconversion responses and use as the basis for COVID-19 serological tests.

We thank Dr. Hideyuki Saya (Keio University School of Medicine) for the critical discussions and suggestions.

This work was supported by the Japan Agency for Medical Research and Development (JP19fk0108110 and JP19fk0108150) and the FUJIFILM Wako Pure Chemical Corp.

The online version of this article contains supplemental material.

Abbreviations used in this article

AUC

area under curve

CoV

coronavirus

COVID-19

coronavirus disease 2019

E

envelope

M

membrane

MERS-CoV

Middle East respiratory syndrome coronavirus

MNT

microneutralization test titer

N

nucleocapsid

PBST

PBS containing 0.1% Tween 20

RBD

receptor-binding domain

RBD-IgG

IgG against RBD

ROC

receiver operating characteristic

RT

room temperature

S

spike

SARS-CoV-2

severe acute respiratory syndrome coronavirus 2

S full

S full length

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K.Y., Y. Yoshida, and Y. Yagura are employees of FUJIFILM Wako Pure Chemical Corp. T.O. is an employee of FUJIFILM Corp. The other authors have no financial conflicts of interest.

Supplementary data