Sensitivity in Detection of Antibodies to Nucleocapsid and Spike Proteins of Severe Acute Respiratory Syndrome Coronavirus 2 in Patients With Coronavirus Disease 2019

Abstract

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), is associated with respiratory-related disease and death. Assays to detect virus-specific antibodies are important to understand the prevalence of infection and the course of the immune response.

Methods: Quantitative measurements of plasma or serum antibodies to the nucleocapsid and spike proteins were analyzed using luciferase immunoprecipitation system assays in 100 cross-sectional or longitudinal samples from patients with SARS-CoV-2 infection. A subset of samples was tested both with and without heat inactivation.

Results: At >14 days after symptom onset, antibodies against SARS-CoV-2 nucleocapsid protein showed 100% sensitivity and 100% specificity, whereas antibodies to spike protein were detected with 91% sensitivity and 100% specificity. Neither antibody levels nor the rate of seropositivity were significantly reduced by heat inactivation of samples. Analysis of daily samples from 6 patients with COVID-19 showed anti-nucleocapsid and spike protein antibodies appearing between days 8 and 14 after initial symptoms. Immunocompromised patients generally had a delayed antibody response to SARS-CoV-2, compared with immunocompetent patients.

Conclusions: Antibody to the nucleocapsid protein of SARS-CoV-2 is more sensitive than spike protein antibody for detecting early infection. Analyzing heat-inactivated samples with a luciferase immunoprecipitation system assay is a safe and sensitive method for detecting SARS-CoV-2 antibodies.

Keywords: COVID-19; SARS-CoV-2; coronavirus; serology.

Figure 1.

Detection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and spike protein in patients with coronavirus disease 2019 (COVID-19). Antibody levels against SARS-CoV-2 nucleocapsid and spike protein were determined in 32 pre-2018 blood donors, 10 patients with suspected COVID-19 (not confirmed with polymerase chain reaction [PCR]) from EvergreenHealth, Kirkland, Washington (EH) and patients PCR positive (PCR+) for COVID-19, including 3 from the University of California, San Diego (UCSD), 13 from the University of Washington (UW), 13 from EH, and 6 from the National Institutes of Health Clinical Center (NIH). Each symbol represents a sample from an individual patient or different time point from an individual patient. Antibody levels are plotted in light units (LU) on a log₁₀ scale. Black circles represent plasma or serum samples obtained >14 days after symptom onset; orange circles, samples obtained ≤14 days after symptom onset; and dashed lines, cutoff levels for determining positive antibody titers, as described in Methods.

Figure 2.

Longitudinal profile of antibodies against nucleocapsid and spike proteins in immunocompetent and immunocompromised patients with coronavirus disease 2019 (COVID-19) from the National Institutes of Health (NIH). Antibody levels were determined in daily blood samples from 6 patients with COVID-19. Three patients (NIH patients 1–3 [NIH-1, NIH-2, and NIH-3]) were immunocompetent (A) and 3 (NIH patients 4–6 [NIH-4, NIH-5, and NIH-6]) were immunocompromised (B). The levels of antibody to the nucleocapsid (black lines) and to spike (blue lines) proteins over time were plotted on the y-axis, using a log₁₀ scale. Time 0 represents the day symptoms appeared; arrows, the time of diagnosis with polymerase chain reaction; dotted lines, cutoff values for determining seropositivity; and red X’s, the day after onset of symptoms that NIH patient 3 died.

Figure 3.

Heat inactivation of plasma or serum samples has no significant impact on detection of nucleocapsid antibodies. A subset (n = 38) of plasma samples from patients with coronavirus disease 2019 (COVID-19) was analyzed, including samples from polymerase chain reaction–positive patients during very early infection (<8 days after onset of symptoms) and at later times after initial infection. Levels of antibody to the nucleocapsid protein were determined using luciferase immunoprecipitation system assays for aliquots of paired unheated and heated plasma or serum samples. Antibody levels were plotted. The horizontal and vertical dotted lines represent cutoff values for seropositivity, and the diagonal line represents theoretically identical antibody levels for heated and unheated samples. Antibody values strongly correlated for heat-treated and unheated samples as shown by the Spearman rank correlation (r_s = 0.92; P < .001); only 1 sample showed a significant decrease with heating.