Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19)

Abstract

Background: The emergence of coronavirus disease 2019 (COVID-19) is a major healthcare threat. The current method of detection involves a quantitative polymerase chain reaction (qPCR)-based technique, which identifies the viral nucleic acids when present in sufficient quantity. False-negative results can be achieved and failure to quarantine the infected patient would be a major setback in containing the viral transmission. We aim to describe the time kinetics of various antibodies produced against the 2019 novel coronavirus (SARS-CoV-2) and evaluate the potential of antibody testing to diagnose COVID-19.

Methods: The host humoral response against SARS-CoV-2, including IgA, IgM, and IgG response, was examined by using an ELISA-based assay on the recombinant viral nucleocapsid protein. 208 plasma samples were collected from 82 confirmed and 58 probable cases (qPCR negative but with typical manifestation). The diagnostic value of IgM was evaluated in this cohort.

Results: The median duration of IgM and IgA antibody detection was 5 (IQR, 3-6) days, while IgG was detected 14 (IQR, 10-18) days after symptom onset, with a positive rate of 85.4%, 92.7%, and 77.9%, respectively. In confirmed and probable cases, the positive rates of IgM antibodies were 75.6% and 93.1%, respectively. The detection efficiency by IgM ELISA is higher than that of qPCR after 5.5 days of symptom onset. The positive detection rate is significantly increased (98.6%) when combining IgM ELISA assay with PCR for each patient compared with a single qPCR test (51.9%).

Conclusions: The humoral response to SARS-CoV-2 can aid in the diagnosis of COVID-19, including subclinical cases.

Keywords: COVID-19; ELISA; antibody; diagnosis; novel coronavirus.

Figure 1.

Cross-reactivity between human plasma against NL63, 229E, OC43, and HKU1 with the N protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). A, Western blot analysis to determine the reactivity of human plasma containing antibodies against NL63, 229E, OC43, and HKU1. Plasma samples were diluted at 1:400 and incubated with the N protein of NL63, 229E, OC43 HKU1, and SARS-CoV, which was loaded at 250 ng/well. The N protein of SARS-CoV-2 was also loaded in each gel to determine the cross-reactivity. B, ELISA showing the reactivity of human plasma against NL63, 229E, OC43, HKU1, and SARS-CoV. Plasma samples were diluted at 1:400 to test the cross-reactivity against the N protein of SARS-CoV-2. The coating amount of N proteins was 10 ng/well. The absorbance (OD) values at 450 nm are shown on the y axis. Abbreviations: Abs, absorbance; ELISA, enzyme-linked immunosorbent assay; OD, optical density; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2.

Figure 2.

Characteristics of SARS-CoV-2 nucleocapsid gene. A, Phylogenetic analysis of the viral nucleocapsid gene. The SARS-CoV-2 is labeled in red in the phylogenetic trees. Other human coronaviruses from a public database are listed as an outgroup. Evolutionary distances were calculated with the neighbor-joining method. B, Amino acid sequence comparison of the nucleocapsid gene of SARS-CoV-2, IPBCAMS-WH-01/2019 (EPI_ISL_402123), compared with SARS-CoV (NC004718).

Figure 3.

Characteristics of plasma antibodies in patients infected with SARS-CoV-2. A, Time of appearance of IgM, IgA, and IgG antibodies to SARS-CoV-2, determined by ELISA of plasma samples obtained from inpatients with SARS-CoV-2 infection. As we only wanted to show the early time points, the plasma samples used for detection of IgM and IgA antibodies were selected from patients who had fever or respiratory infectious symptoms within 7 days. The plasma samples used for detection of IgG antibodies were selected from all the patients whose IgG antibodies were positive. B, Levels of IgM, IgA, and IgG antibodies against SARS-CoV-2 in plasma samples after symptom onset. Antibody titers are expressed as GMTs. Abbreviations: ELISA, enzyme-linked immunosorbent assay; GMT, geometric mean titer; Ig, immunoglobulin. *P <.05.

Figure 4.

IgM ELISA to diagnose patients infected with COVID-19. A, IgM antibody detection using ELISA in patients who were either PCR positive (82 cases) or who tested negative for the virus by qPCR (58 cases). Red indicates IgM positive while blue indicates IgM negative. B, Fitted curve of the positive detection by PCR and IgM or IgG ELISA on different days after symptom onset. The fitted curves were created by the Fit Spline program of Graphpad Software. The 95% confidence intervals are shown for each curve. The dots in the upper panel represent positive rates of PCR and IgM or IgG ELISA at each time point. The lower table shows the number of samples that tested positive at each time point. The first intersection of the real-time PCR method and IgM ELISA was found to be at 5.5 days. C, The positive detection rate of cases using the PCR method versus PCR plus IgM ELISA assay. Abbreviations: COVID-19, coronavirus disease 2019; ELISA, enzyme-linked immunosorbent assay; Ig, immunoglobulin; PCR, polymerase chain reaction; qPCR, quantitative polymerase chain reaction.