Humoral Immune Response to SARS-CoV-2

Pauline H. Herroelen, MSc; Geert A. Martens, MD, PhD; Dieter De Smet, MD; Koen Swaerts, MSc; An-Sofie Decavele, MSc


Am J Clin Pathol. 2020;154(5):610-619. 

In This Article

Materials and Methods


Serum samples were obtained from the following cohorts: (1) hospitalized COVID-19 patients—105 serum samples obtained at different time points from 71 patients with PCR-confirmed SARS-CoV-2 infection and admitted for severe COVID-19 pneumonia from March 1 to April 27, 2020, at our tertiary AZ Delta General Hospital in Roeselare, Belgium; and (2) patients with paucisymptomatic SARS-CoV-2 infections—66 serum samples from 64 health care workers with a SARS-CoV-2 infection, PCR-confirmed after developing fever and World Health Organization (WHO)-listed COVID-19 symptoms. These patients were home-quarantined without need for hospitalization. The study was approved by the AZ Delta ethical committee with a waiver of informed consent from the hospitalized COVID-19 patients and with written informed consent from participants with paucisymptomatic SARS-CoV-2 infections.

Cross-reactivity was evaluated on a panel composed of 57 prepandemic serum samples obtained from patients with PCR-confirmed infection by other HCoV respiratory viruses (HCoV 229E, n = 1; HCoV HKU1, n = 3; HCoV OC43, n = 2; HCoV OC43 + adenovirus, n = 1), other pathogens and viruses (n=42), or presence of autoimmune antibodies (n = 8). Serum samples from patients with other HCoV infections ranged from 0 to 39 days after PCR positivity Table 1.

SARS-CoV-2 Serology Assays

All serology assays were used according to manufacturers' protocol using cutoffs specified in package inserts as detailed below.

Rapid Tests. COVID-19 IgG/IgM Rapid Test (Zhejiang Orient Gene Biotech) is a solid phase immunochromatographic assay for qualitative detection of IgM and IgG antibodies to recombinant N and S proteins. Innovita 2019-nCoV Ab Test (Innovita Biological Technology) is a colloidal gold lateral flow assay for qualitative detection of IgM and IgG antibodies to undisclosed SARS-CoV-2 epitopes. Rapid tests were considered positive if a line was observed for IgM, IgG, or both. Color intensity was not evaluated.

ELISA. Wantai SARS-COV-2 Ab ELISA (Beijing Wantai Biological Pharmacy Enterprise) is a double-antigen sandwich immunoassay for qualitative detection of all antibody isotypes (IgM, IgA, IgG) against RBD domain of S1 protein. Samples with a cutoff ratio (absorbance of the sample at 450 nm divided by 0.19) higher than 0.9 were considered positive (classifying gray zone results with cutoff ratio 0.9–1.1 as positive). Three indirect ELISAs from EUROIMMUN were tested: Anti-SARS-CoV-2 IgG and IgA assays for semiquantitative detection of IgA and IgG antibodies against S1 protein and Anti-SARS-CoV-2-NCP(IgG) assay for semiquantitative detection of IgG against N protein. (cutoff of 0.8 units, classifying gray zone results of 0.8–1.1 units as positive). All ELISAs were tested using the PhD system (Version EIA 0_16, Bio-Rad Laboratories).

Chemiluminescent Immunoassays. Elecsys Anti-SARS-CoV-2 assay for cobas e601 module (Roche Diagnostics) is a double-antigen sandwich assay for qualitative detection of all antibody isotypes (IgM, IgA, IgG) against N protein (cutoff of 1 cutoff index). LIAISON SARS-CoV-2 S1/S2 IgG (DiaSorin) is an indirect chemiluminescent immunoassay for quantitative detection of IgG antibodies against S1/S2 proteins (cutoff of 12 arbitrary units [AU]/mL, classifying gray zone results of 12–15 AU/mL as positive).

SARS-CoV-2 PCR. This was done using the Allplex 2019-nCoV assay (Seegene) for E/N/RdRP genes on nasopharyngeal swabs.

Statistical Analysis

Statistical analyses were performed using MedCalc (version 12.2.1). Sensitivities for detection of presence of SARS-CoV-2 antibodies were evaluated on samples obtained from SARS-CoV-2 PCR-positive patients as (1) total fraction of samples showing detectable antibodies and (2) by comparing each individual assay vs consensus outcome obtained by majority of all assays evaluated in this study. χ2 test was used for comparing proportions for categorical variables. Not normally quantitative variables are expressed as medians (interquartile range [IQR]), and Mann-Whitney test was used to test for statistical differences between various timeframes after onset symptoms. Differences were considered statistically significant if P < .05. Kinetics of seroconversion in individual patients in Figure 1 were fitted to a scale from −1 to +1, with 0 representing each assays cutoff by subtracting each assay's cutoff from its raw data signals, and dividing its absolute value by highest (lowest) cutoff-corrected signal for that assay obtained in our data set for positive (negative) samples.

Figure 1.

Kinetics of seroconversion in critically ill COVID-19 patients. The upper left panel shows the average kinetics of seroconversion in 13 intensive care unit patients. The other panels show the kinetics in 8 individual patients for whom 3 or more data points were available. Graphs represent for each of the indicated serology tests the normalized signal over time, fitted to a scale from –1 to +1 with 0 (black line) representing the assays' cutoff, as described in the Statistical Analysis section.