Clinical significance of SARS‐CoV‐2‐specific IgG detection with a rapid antibody kit for COVID‐19 patients

Abstract Background The longitudinal observation of the detection of antibody responses to SARS‐CoV‐2 using antibody kits during the clinical course of COVID‐19 is not yet fully investigated. Objectives To understand the significance of the detection of anti‐SARS‐CoV‐2 antibodies, particularly IgG, using a rapid antibody kit, during the clinical course of COVID‐19 patients with different severities. Methods Sixty‐three serum samples from 18 patients (5 asymptomatic and 13 symptomatic patients) were retrospectively examined using a commercial SARS‐CoV‐2 IgM/IgG antibody kit. PCR positivity of patient samples was also examined as a marker of current SARS‐CoV‐2 infection. Results IgG antibodies were detected in all cases in this study. The IgG detection rates reached 100.0% in samples collected on day 13 or later. IgG seropositivity after an initial negative status was observed in 13 patients (3/5 asymptomatic and 10/13 symptomatic cases). Interestingly, the persistence of both PCR and IgG positivity was detected in seven cases, of which three were asymptomatic. The longest overlap duration of the PCR and IgG positivity was 17 days in asymptomatic status. Conclusions SARS‐CoV‐2‐specific IgG production can be detected in all infected individuals, using a rapid antibody kit, irrespective of clinical status. However, these findings suggest that, in some infected individuals, particularly those with asymptomatic status, the presence of virus‐specific IgG antibodies does not imply prompt viral clearance.


| INTRODUC TI ON
The novel coronavirus disease , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread worldwide, and the World Health Organization (WHO) declared it a pandemic, on March 11, 2020. In Japan, the COVID-19 outbreak has been accelerating since April 2020. A PCR assay of nasal and pharyngeal swab samples is generally used as the standard method for COVID-19 diagnosis. On the other hand, serological testing based on the host's antibody responses to SARS-CoV-2 is being investigated to clarify how useful it is in the category of COVID-19. 1,2 Rapid antibody detection kits, which can detect antibodies in human blood within a short time, are expected to be applied in the various situations of COVID-19 management, and many, such kits are already used in the market. There is an ongoing verification of the accuracy of these antibody kits, in terms of sensitivity and specificity. 3,4 However, the longitudinal observation of the detection of antibody responses to SARS-CoV-2, using antibody kits, during the clinical course of COVID-19, has not been fully investigated, although some studies have been reported. 5,6 We attempt to discuss the significance of the detection of anti-SARS-CoV-2 antibodies, particularly IgG. In this study, we examined the detection of antibodies against SARS-CoV-2 and the relationship between seroconversion and PCR positivity, using an antibody kit in COVID-19 patients with different degrees of severity. We also discuss the utility of a rapid antibody kit in the clinical and epidemiological setting of COVID-19, based on the results of this study.

| Patients and sample collection
This retrospective study was conducted using samples obtained from patients admitted to the Kyushu University Hospital, Fukuoka, Japan. All patients had been diagnosed with COVID-19 before admission, on the basis of a positive result from the real-time PCR assay of nasal and pharyngeal swab specimens that was performed by the Japanese Institute of Health according to the manual for the detection of pathogen 2019-nCoV. 7

| Study definitions
The presence of fever was defined as an axillary temperature of 37.5°C or higher. 8,9 The severity of respiratory symptoms was graded as absent, mild, moderate, or severe. 9 The patients were classified as symptomatic if they had at least a temperature higher than 37.5°C and/or moderate-to-severe respiratory symptoms. 9 The symptomatic status was evaluated at the time of hospital admission.
Asymptomatic patients were those who did not meet the symptomatic condition, neither before nor after admission. The severity of the symptomatic status was categorized as mild, severe, or critical.
Mild symptomatic cases were characterized by blood oxygen saturation ≥93% and no oxygen requirement. Severe cases included blood oxygen saturation ≤92% and requirement of oxygen, delivered through a nasal cannula or an oxygen mask. Critical cases were those requiring mechanical ventilation for oxygen supply. For symptomatic patients, the day of symptoms onset (day 1) was considered as the beginning of the symptomatic status, as defined above. Day 1 for asymptomatic patients began on the first day of PCR positivity.

| RE SULTS
A total of 63 samples from 18 patients were collected and analyzed in our study. Virus-specific IgM antibodies were not detected in any of the samples, except for one (case 12), during the collection period ranging from day 2 to day 33 after symptoms onset, regardless of the number of days (1/63 samples, 1.6%) ( Table 1).
Virus-specific IgG antibodies were detected in all cases during the collection period (18/18 cases, 100.0%) ( Table 2). IgG antibodies were not detected in samples collected by day 6 after symptoms onset (0/8 samples, 0.0%). The detection rate of IgG was 41.4% (7/17 samples) in samples collected from day 7 to 9 after symptoms onset.
Of these, four samples were from asymptomatic patients. The IgG detection rates increased to approximately 70% during days 10-12 after symptoms onset and reached 100.0% in samples collected on day 13 or later after symptoms onset.  Therefore, it is intriguing that the prolonged overlap duration of PCR and IgG positivity was observed in asymptomatic patients in this study. This is a new finding that has not been indicated yet, to our best knowledge. In patients with few symptoms, appropriate immune responses might not have occurred, possibly due to the differential quantity or quality of the immune responses. The detection of antibodies to the nucleocapsid protein of SARS-CoV-2 was examined in this study. It may be attractive to investigate antibodies to other proteins of the virus, particularly the spike protein, which is the main antigen that elicits neutralizing antibodies. We need to further investigate the immunological mechanisms by which the delay of viral elimination occurs, even after the production of SARS-CoV-2-specific IgG antibodies.

| D ISCUSS I ON
The detection rate for anti-SARS-CoV-2 IgM antibodies in the kit in this study was extremely low. Virus-specific IgM antibodies are generally produced faster than IgG antibodies after viral exposure.
Other assays for anti-SARS-CoV-2 IgM and IgG antibodies reported that it was difficult to selectively detect virus-specific IgM before IgG within 7 days after symptoms onset. 11  This study had some limitations. First, the sample size was small.
Although the findings obtained from the asymptomatic patients were interesting, only five asymptomatic subjects were enrolled.
Second, the study was retrospective, and therefore, it was not possible to evaluate the precise timing of IgG seroconversion or PCR negativity because of the lack of a prospective sample collection.
Third, the presence of anti-SARS-CoV-2 antibodies detected by the kit used in the study should be verified through quantification assays. Nevertheless, the findings of this study are informative. They status that viral clearance after IgG production is not similar and that a delay of viral elimination, even after IgG production, can be observed, particularly in some individuals with asymptomatic status.

ACK N OWLED G EM ENTS
We thank all doctors for participating in this study.

CO N FLI C T O F I NTE R E S T
We have no financial conflicts of interest to declare.