The novel coronavirus (SARS-CoV-2) is a new virus responsible for an outbreak of respiratory illness known as COVID-19, and now it is a global pandemic in more than 215 countries [
Based on the diagnosis experience of many clinical cases, the detection of novel coronavirus antibody can be used as an auxiliary diagnosis of novel coronavirus pneumonia [
Chemiluminescence immunoassay (CLIA) has been developed as an effective combination of immunoassay and chemiluminescence system [
This study enrolled a total of 178 patients who visited Huangshi Central Hospital in Hubei Province, China, between January and February 2020. The patients included 91 males (51.1%) and 87 females (48.9%) with a mean age of 54.3 years (ranging from 2 months to 94 years). Among them, the SARS-CoV-2 group had 68 patients, 36 males and 32 females (ranging from 30 years to 90 years); the suspected group had 9 patients, 7 males and 2 females (ranging from 2 months to 64 years); and the negative group had 101 patients, 48 males and 53 females (ranging from 2 years to 94 years). This study is in compliance with ICC clinical trial specifications and the Helsinki Declaration.
Serum was collected from all patients. Serum SARS-CoV-2 IgG and IgM were tested by CLIA kits and the iFlash 3000 fully automated CLIA analyzer obtained from Shenzhen YHLO Biotech Co., Ltd (China). In brief, serum was separated by centrifugation at 2500 g for 5 min within 12 hours of collection. The magnetic beads of these CLIA assays are coated with two antigens of SARS-CoV-2 (nucleocapsid protein (N protein) and spike protein (S protein)). SARS-CoV-2 IgM/IgG titers (in arbitrary units, AU/ml) were calculated automatically by the CLIA analyzer based on relative light units (RLU), and the viral antibody titer was positively associated with RLU. The cutoff values for positive SARS-CoV-2 IgM and IgG are both 10 AU/ml.
RT-PCR was used to detect open reading frame 1ab (ORF1ab) and nucleocapsid protein (N) in the SARS-CoV-2 genome. CT value interpretation of test results is based on the instruction from the manufacturer. Confirmation of positive COVID-19 is based on at least one target-specific RT-PCR-positive result of ORF1ab and N genes of SARS-CoV-2 in the same specimen.
Statistical analysis was performed using SPSS 19.0 statistical software (IBM SPSS, Chicago, IL, USA). The kappa coefficient was calculated. Kappa ≥0.75 indicates good consistency, 0.75 ≥ kappa > 0.4 for medium consistency, and kappa <0.4 for poor consistency.
The specificity and sensitivity of the CLIA test kits were calculated according to the following equations:
Samples from both NAT-negative patients (suspected group, 9 subjects) and other diseases’ population (control group, 101 subjects) were used to assess the clinical specificity of the assay (Table
Clinical specificity of SARS-CoV-2 IgM and SARS-CoV-2 IgG.
Number of samples | SARS-CoV-2 IgM | SARS-CoV-2 IgG | |||||||
---|---|---|---|---|---|---|---|---|---|
N | P | Clin Spe (%) | 95% CI | N | P | Clin Spe (%) | 95% CI | ||
Suspected group | 9 | 9 | 0 | 100.00 | (70.1%, 100.0%) | 9 | 0 | 100.00 | (70.1%, 100.0%) |
Control group | 101 | 100 | 1 | 99.01 | (94.6%, 99.8%) | 97 | 4 | 96.04 | (90.3%, 98.4%) |
N: negative; P: positive; Clin Sep: clinical specificity.
Samples from 68 SARS-CoV-2-infected patients (confirmed with RT-PCR) were used to evaluate the clinical sensitivity of the assays (Table
Clinical sensitivity of SARS-CoV-2 IgM and SARS-CoV-2 IgG.
Days | Number of samples | SRAS-CoV-2 IgM | SRAS-CoV-2 IgG | ||||||
---|---|---|---|---|---|---|---|---|---|
N | P | Clin Sen (%) | 95% CI | N | P | Clin Sen (%) | 95% CI | ||
<7 days | 12 | 3 | 9 | 75.00 | (46.8%, 91.1%) | 2 | 10 | 83.33 | (55.2%, 95.3%) |
7–14 days | 25 | 3 | 22 | 88.00 | (70.0%, 95.8%) | 0 | 25 | 100.00 | (86.7%, 100.0%) |
>14 days | 31 | 2 | 29 | 93.55 | (79.3%, 98.2%) | 0 | 31 | 100.00 | (89.0%, 100.0%) |
Total | 68 | 8 | 60 | 88.24 | (78.5%, 93.9%) | 2 | 66 | 97.06 | (89.9%, 99.2%) |
Days: days since the onset of symptoms; N: negative; P: positive; Clin Sen: clinical sensitivity.
The comparison between SARS-CoV-2 IgM/IgG antibody test and NAT of 178 patients is shown in Table
Comparison of SARS-CoV-2 IgM/IgG antibody detection and SARS-CoV-2 nucleic acid detection.
IgM/IgG antibody | ||||||
---|---|---|---|---|---|---|
Positive | Negative | Total | Positive predictive value of NAT (%) | Negative predictive value of NAT (%) | ||
Nucleic acid | Positive | 67 | 1 | 68 | 100.00 | |
Negative | 5 | 105 | 110 | 96.36 | ||
Total | 72 | 106 | 178 | |||
Positive predictive value of antibody test | 93.06 | |||||
Negative predictive value of antibody test | 99.06 |
Novel coronavrius disease is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [
Since February 2020, several SARS-COV-2 IgM and IgG antibody immunoassay kits have been developed in China. Antibody detection is a new detection method for SARS-CoV-2, so the clinical specificity and sensitivity of such tests must be carefully validated [
In COVID-19 cases, the clinical sensitivity of SARS-CoV-2 IgM detection was 88.23%, while the clinical sensitivity of SARS-CoV-2 IgG detection was 97.06%. The CLIA system can simultaneously detect 150–300 clinical samples, which is a good tool for screening and diagnosis of the novel coronavirus pneumonia caused by SARS-CoV-2. Our study results showed that the combined detection of SARS-CoV-2 IgM and IgG antibodies is an effective tool to improve the diagnostic sensitivity and specificity and reduce the chance of false-negative NAT results. We demonstrate that the antibody detection can be used as one of the effective methods of COVID-19 clinical detection.
In the NAT-confirmed group, serum from 68 COVID-19 cases was tested for SARS-CoV-2 IgM and IgG. SARS-CoV-2 IgM antibodies can be detected in 75.00% of patients before 7 days since the onset of the symptoms, and the positive rate reached to 88.00% on the period of 7–14 days and then increased to 93.55% after 14 days. The positive rate of SARS-CoV-2 IgG was 83.33% before 7 days since the onset of symptoms and reached to 100.00% on 7–14 days and remained 100% after 14 days. In general, the immune response to infection by pathogenic microorganisms is first expressed as an increase in the IgM antibody titer and then a rapid decrease until it disappears, while the IgG antibody titer normally increased in the middle and late stages of the infection, and it can be positive for a long time even after recovery. According to the results of this study, the positive rate of IgM in SARS-CoV-2-infected patients is lower than that of IgG because most of the infected patients were in the middle stage of infection or in the recovery stage. Interestingly, we have observed a phenomenon that SARS-CoV-2 IgM and IgG antibodies developed almost simultaneously, and this observation is consistent with some recent studies [
We found false-negative results for IgM/IgG in the NAT group. There might be three reasons: first of all, false-negative results may be due to low antibody titer. When IgM and IgG titers are below the detection limit, the test result might be negative. Secondly, the difference in individual immune response and antibody production could be another reason for the false-negative results in COVID-19 patients. The last reason might be that IgM antibody might decrease or even disappear after 15 days. In each individual case, it is difficult to know exactly when or how long the patient has been really infected, and someone might have IgM titer below the detection limit and not detectable. In the joint detection of SARS-CoV-2 IgM and IgG, there was only one negative patient (male, 77 years old) who had respiratory failure, chronic obstructive pulmonary disease, coronary atherosclerosis, acute myocardial infarction, and heart failure with SARS-CoV-2 infection. In the control group, 5 cases were positive for antibody detection (1 case for IgM and 4 cases for IgG). The results suggested that the patients who had some other diseases, including tumors, leukemia, diabetes, hypertension, coronary atherosclerosis, bronchitis, or lung infections, might be more susceptible to be infected by SARS-CoV-2 and led to positive antibody detection. Also, there might be false-negative nucleic acid or recovered/mild/asymptomatic patients with SARS-CoV-2. In addition, it is well known that the positive and negative predictive values are not only intrinsic to the test but also depend on the prevalence [
Our study also has some limitations. For example, we did not investigate the cross-reaction with other pathogens (e.g., hCoV-NL-63 or others), MERS-CoV, SARS-CoV, and some auto-antibodies that could cause interference for immunoassay. Also, we did not perform dynamic monitoring of the change of antibody titer for in-depth study.
Overall, testing SARS-CoV-2 IgG and IgM by the CLIA method is convenient for sampling, and it has high efficiency. The results of this study indicated that combined detection of serum IgM and IgG antibodies to SARS-CoV-2 had better sensitivity and specificity compared with single IgM or IgG antibody testing. Therefore, the serological test results can be used as an effective diagnostic tool for SARS-CoV-2 infection. It can also be used as an efficient supplement of RNA detection for confirmation of SARS-CoV-2 infection in clinics, hospitals, and accredited scientific laboratories.
Severe acute respiratory syndrome coronavirus 2
Nucleic acid test
Chemiluminescence immunoassay
Immunoglobulin M
Immunoglobulin G.
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
All the SARS-CoV-2 IgG and IgM CLIA kits used in this study were kindly supplied by the manufacturer, namely, YHLO Biotech (Shenzhen, China).
The authors declare that they have no conflicts of interest.
Fang Hu analyzed the data, drafted the article, and contributed to study design. Xiaoling Shang and Changliang Zhang contributed to data gathering. Meizhou Chen contributed to study design, editing, and revising the paper. All authors read and approved the final manuscript.