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. 2022 Aug 16:2022:7754329.
doi: 10.1155/2022/7754329. eCollection 2022.

Diagnostic Performance of Three ELISAs for Detection of Antibodies against SARS-CoV-2 in Human Samples

Cássio Meira  1   2 , Dahara Silva  1   2 , Ivanilson Santos  2 , Breno Barreto  2 , Vinícius Rocha  1   2 , Emanuelle Santos  1 , Bruna Dos Reis  2 , Afrânio Evangelista  1 , Ricardo Ribeiro Dos Santos  1   2 , Bruna Machado  1 , Guilherme Ribeiro  2   3 , Roberto Badaró  1 , Milena Soares  1   2
Affiliations

Diagnostic Performance of Three ELISAs for Detection of Antibodies against SARS-CoV-2 in Human Samples

Cássio Meira et al. ScientificWorldJournal. .

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that causes coronavirus disease 2019 (COVID-19) is a disease with a high rate of transmission. Serological tests are important to perform surveys and to determine the immunological status of the population. Based on this, we evaluated three enzyme-linked immunoassays (ELISAs) using different antigens from SARS-CoV-2 in a cohort of 161 patients. The performance of the ELISA developed for immunoglobulin G (IgG) measurement against SARS-CoV-2 was evaluated based on sensitivity, specificity, and accuracy. We found specificities of 0.98, 0.98, and 0.99 and sensitivities of 0.99, 0.91, and 0.87 for the nucleocapsid (N) protein, spike protein, and receptor binding domain (RBD) fraction, respectively. The accuracy assessment indicated the N protein (accuracy = 0.98) as the antigen most likely to give a correct diagnosis. Overall, the antibody responses were present for all three proteins in subjects with confirmed SARS-CoV-2 infections, showing a similar pattern of antibody production for different antigens. In summary, these highly sensitive and specific ELISAs, with a more competitive price, appear to be a valid approach for the serodiagnosis of COVID-19.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Figures

Figure 1
Figure 1
Flowchart of the in-house ELISAs developed. ELISA plates were coated with 50 μL of the different antigens (N, S, or RBD fraction) at 2 µg/mL. After 16 hours, the plates were washed with PBS and blocked with a solution of PBS containing 3% non-fat milk and 0.05% Tween 20 for 1 hour. Next, the serum samples were diluted 1 : 50 in PBS containing 1% nonfat milk and 0.05% Tween 20 and added to plates coated with SARS-CoV-2 antigens. Following 2 hours of incubation, the plates were washed and incubated with horseradish peroxidase-labeled anti-human IgG secondary antibody (1 : 5000 dilution in PBS containing 1% nonfat milk and 0.05% Tween 20). The plates were then washed following 60 min incubation at 37°C, and 3,3′,5,5′-tetramethylbenzidine substrate was added. Two min later, stop buffer was added, and the absorbance values were measured at 450 nm wavelength using a microplate reader.
Figure 2
Figure 2
ELISA performed with different severe acute respiratory syndrome (SARS-CoV-2) proteins used as antigens. (a–c) IgG anti-SARS-CoV-2 detected by ELISA using N (a), S (b), and RBD (c) antigens. The cutoff was set as the mean plus two standard deviations of the healthy control samples. The correlations of the ELISA values between N and S (d), N and RBD (e), and S and RBD (f) antibody values. The Pearson correlation coefficient (r) was used to measure the strength of the correlation between the ELISA results performed with different antigens. The results are from one experiment of the three experiments performed.
Figure 3
Figure 3
Heatmap showing the average reactivities to the three antigens in individual patients. The heatmap was constructed using only the 69 patients with a positive diagnosis for COVID-19 by RT-qPCR, and the samples were collected between 15 to 30 days after a positive molecular diagnosis. The absorbance values found for each protein were normalized based on the cutoff point found for each protein (N, S, and RBD fraction). The color gradient bar represents lower reactivity against antigens in dark blue and higher reactivity against antigens in dark red.
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