Development and comparative evaluation of SARS-CoV-2 S-RBD and N based ELISA tests in various African endemic settings

Abstract

Management of the COVID-19 pandemic relies on molecular diagnostic methods supported by serological tools. Herein, we developed S-RBD- and N- based ELISA assays useful for infection rate surveillance as well as the follow-up of acquired protective immunity against SARS-CoV-2. ELISA assays were optimized using COVID-19 Tunisian patients’ sera and prepandemic controls. Assays were further validated in 3 African countries with variable endemic settings. The receiver operating curve was used to evaluate the assay performances. The N- and S-RBD-based ELISA assays performances, in Tunisia, were very high (AUC: 0.966 and 0.98, respectively, p < 0.0001). Cross-validation analysis showed similar performances in different settings. Cross-reactivity, with malaria infection, against viral antigens, was noticed. In head-to-head comparisons with different commercial assays, the developed assays showed high agreement. This study demonstrates, the added value of the developed serological assays in low-income countries, particularly in ethnically diverse populations with variable exposure to local endemic infectious diseases.

1. Introduction

The emergence and pandemic spread of the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus (SARS-CoV) 2 [1], has led to an unprecedented global public health crisis. SARS-CoV-2 is the seventh virus of the Coronaviridae family that can infect humans and the third zoonotic coronavirus after SARS-CoV and the middle east respiratory syndrome coronavirus (MERS-CoV), to cross the species barrier and cause severe respiratory infections in humans. Coronavirus particles contain 4 main structural proteins, namely the spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins, all of which are encoded within the 3′ end of the viral genome [2].

The spike and the nucleocapsid proteins are highly immunogenic. The trimeric S protein is the first virus component that binds SARS-CoV-2 viral particles to host cell receptors. This interaction occurs through the receptor-binding domain (RBD) of S1 subunit and triggers via the S2 subunit, the fusion of the virus to host membranes. The N protein, the most abundant viral protein, is a structural component of the helical nucleocapsid, which plays an important role in viral pathogenesis, replication, and RNA packaging [3].

Several serological assays have been developed to detect antibodies to the S or S-RBD protein as well as the N protein in patients with COVID-19 [[4], [5], [6], [7], [8]]. Serological testing is a key method for seroprevalence studies at the populational level [9]. Since SARS-CoV-2 continues to spread around the globe, it is crucial to understand the duration and nature of immunity raised in response to infection and to gain information regarding the duration of the protection generated by different vaccines. Thus, many efforts have been made to develop sensitive and specific immunoassays including the development of lateral flow chromatographic or ELISA assays. Access at a lower cost to validated in-house tests which do not require heavy equipment can be of considerable benefit in countries with limited resources.

Herein, we report the development and evaluation of low-cost SARS-CoV-2 S-RBD and N-based in-house ELISA assays at a lower cost in different African settings with variable endemicity of several chronic infections that may induce cross-reactivity and interfere with the assay performance.

2. Materials and methods

2.1. Study population and serum sampling

2.1.1. Participants from Tunisia

The Tunisian cohort consisted of a total of 108 individuals with COVID-19: 45 patients admitted at Abderrahmane Mami hospital (Ariana) and 63 health workers from Charles Nicolle hospital and Institut Pasteur de Tunis (50 Males; 58 Females; aged 25–85 years, median 50 years). Blood was collected from patients within the first 6 weeks after the onset of illness (n = 17 for days 0–7, n = 29 for days 8–14, n = 13 for days 15–28, n = 49 for days 29–42). The serum or plasma was separated and stored at -80 °C until use. Control serum samples (n = 72) from prepandemic sera were collected before the pandemic (between 2013 and 2018). The study protocol was approved by the Institut Pasteur de Tunis ethical committee (2020/21/I/LR16IPT/V2). Patients were included after written informed consent. Noninclusion criteria were as follows: the presence of a mental handicap, pregnant women, and patients on immunosuppressive therapy.

2.1.2. Participants from Senegal

In Senegal, a total of 173 plasma or serum samples were included in the study. The COVID-19 positive cohort includes 67 samples collected from hospitalized patients within the first 6 weeks after the onset of illness (n = 48 for days 15–28 and n = 19 for > 28). COVID-19 cases were confirmed by RT-PCR and samples were collected at 3-day intervals until viral clearance was confirmed with 2 successive PCR negative results. In addition, 106 serum/plasma samples, obtained from COVID-19 negative individuals (sampling performed before September 2019) and selected from a well-documented biobank of Institut Pasteur de Dakar, were included. This prepandemic panel of samples was constituted of blood samples collected from people living in malaria endemic areas and with (n = 37) or without (n = 16) IgG against Plasmodium falciparum crude extract antigen. It also included samples from patients diagnosed by molecular or serological assays for various viral diseases including human immunodeficiency virus (n = 20), Influenza A/B (n = 17), Chikungunya (n = 9), and Yellow fever (n = 2). Patients harboring rheumatoid factor proteins associated with arthritis (n = 5) were also included in this panel of COVID-19 negative controls. To further investigate possible antibody cross-reactivity due to Plasmodium antigens, all the non-COVID-19 sera were also tested in malaria serology and divided into 2 groups: malaria IgG positive (n = 72) et malaria IgG negative (n = 34). All plasma or serum samples were stored at – 20 °C until used. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Senegalese National Ethics Committee for research in health (approval N° 00000068/MSAS/CNERS/Sec, 10 April 2020).

2.1.3. Participants from Morocco

The Moroccan cohort consisted of a total of 112 sera from 95 patients with COVID-19. These patients were recruited as part of a longitudinal study that started on March 18, 2021, in the marquees of 2 hospitals in Casablanca: Moulay Youssef Regional Hospital and Mohamed Bouafi Hospital. Study participants were adults, unvaccinated against COVID-19 with a positive RT-qPCR test, symptomatic who provided informed consent: (38 males; 57 females; age 18–87 years, median 52 years). Blood was collected from patients within the first 9 weeks after disease onset (n = 89 for days 15–20, n = 13 for days 30–35, and n = 10 for days 60–65). Serum or plasma was separated and stored at -80 °C until use. Control serum samples (n =113) from the general population were collected before the pandemic (between 2016 and 2017). The study protocol was approved by the ethics committee of the Mohammed VI University of Health Sciences of Casablanca (CERB/UM6SS).

2.1.4. Participants from Madagascar

Samples from the Malagasy cohort were collected during the first few cases of investigations of COVID-19 and their contacts in Antananarivo, Madagascar (FFX samples). Whole blood samples were collected from individuals who had positive PCR tests. Plasmas samples were stored at -20 °C until analysis. Ninety samples were analyzed in this study. Ethical approvals for this study and the use of blood collected from this cohort were given by the Biomedical Research Committee of the Ministry of Public Health in Madagascar (no. 058/MSANP/SG/AGMED/CERBM, March 30, 2020). Malagasy prepandemic samples were obtained from a cross-sectional survey performed in 2015 and approved by the Ethics Committee of Biomedical Research of the Ministry of Public Health of Madagascar.

2.2. Recombinant proteins production and purification

Recombinant N protein was produced in Escherichia coli BL21 (DE3) using the pETM11/N-nCov-(His)6-Nter plasmid as previously described [10]. In brief, 1 selected colony was suspended and cultured in an LB medium. When OD₆₀₀ reached 1, isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.2 mM and incubation was continued for 3 hours. Subsequently, the bacterial cells were lysed by sonication in the urea lysis buffer (8 M Urea, 100 mM NaCl, 20 mM Tris-HCl, and 10 mM Imidazole). The recombinant N protein was then purified using affinity chromatography followed by gel filtration. Protein expression and purification were monitored by SDS-polyacrylamide gel electrophoresis.

For proper post-translational modifications (i.e., glycosylation, and correct folding), the S protein and its RBD fragment require to be expressed in eukaryotic systems [[11], [12], [13]]. The S-RBD (residues 319–541) of the SARS-CoV-2 spike protein [14] was cloned into a customized pFastBac vector. Recombinant bacmid DNA was generated using the Bac-to-Bac system following the manufacturer's instruction (Life Technologies, Thermo Fisher Scientific).

The recombinant bacmid DNA was transfected into S-f9 cells. S-f9 cells were then infected with the recombinant baculoviruses at an MOI of 3. Seventy-two hours postinfection, S-RBD protein was collected and then purified, after a concentration step, by AKTӒ purifier system (GE Healthcare Life Sciences, Uppsala, Sweden) using affinity His Trap Flow fast 5 mL column.

The quality control of the produced recombinant proteins was performed using several tests including western blot and MALDI-TOF.

2.3. Indirect ELISA for IgG antibodies to S-RBD and N SARS-CoV-2 proteins

Ninety-six–well ELISA plates were coated overnight with recombinant proteins in phosphate-buffered saline (PBS) (50 ng for N and 100 ng for S-RBD in a volume of 50 µL per well). After washing 3 times with PBS–0.1% Tween 20 (PBS-T), plates were then incubated with 4% bovine serum albumin in PBS-T for 1 hour at 37 °C for the blocking step. After 3 washes, 50 µL of diluted sera (1:200 for the N-based and 1:400 for the S-RBD-based assays) in PBS-T was added and incubated for 2 hours at 37 °C. Plates were then washed 6 times, and incubated with 8,000-fold diluted peroxidase-conjugated goat anti-human IgG (Sigma, AG029) for 1 hour at 37°C. After 6 washes, plates were revealed by adding 50 µLof horseradish peroxidase (HRP) chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (BD Biosciences, 555214). After 10 min incubation, the reaction was stopped by adding 50 µL of sulfuric acid (2N) and ODs were measured at 450/630 nm.

2.4. Statistical analysis

Receiving operator characteristic curves analysis was performed to determine the accuracy of the in-house ELISA assays considering RT- PCR as the gold standard to define SARS-CoV-2 positivity status. Specificity and sensitivity with their 95% confidence intervals were determined using the optimal cut-off values. We further studied the agreement between the developed in-house ELISA assays and the commercial kits by estimating the Kappa coefficient.

Statistical analyses using nonparametric tests were performed using Graphpad Prism (versions 5 and 8) and P < 0.05 was considered statistically significant.

3. Results

3.1. Proteins expression and purification

3.1.1. Recombinant SARS-CoV-2 nucleoprotein production

The N recombinant protein was produced in E. coli BL21 (DE3) using the His-Tag cloning system and pETM11 vector. Several parameters, namely temperature variation, aeration, cell density, and induction time were adjusted to optimize the expression conditions of the recombinant protein (data not shown). The recombinant protein expression was induced when reaching a cell density of 1 OD at 600 nm for 3 hours after the addition of 0.2mM isopropyl-β-D-thiogalactopyranoside. A major band with molecular weight around 46KDa by 12% SDS-PAGE was detected as a recombinant N protein (Fig. 1 A). The protein was then purified using affinity chromatography followed by a gel filtration step to obtain highly purified protein. Quality control assays were performed including MALDI-TOF ( data not shown ).

Fig. 1.

Recombinant proteins expression and purification. Recombinant N and S-RBD proteins were produced in E.coli BL21 (DE3) and Sf9 cells, respectively. (A) SDS-PAGE analysis of the N protein purified using Ni-NTA affinity chromatography followed by gel filtration Sephadex G75; (B) Silver staining of recombinant S-RBD produced using baculovirus/S-f9 expression system and purified using Ni-NTA affinity followed by gel filtration superdex 200; (C) Western blotting using a monoclonal anti-Histidine-tag antibody to detect recombinant S-RBD protein.

3.1.2. Recombinant SARS-CoV-2 S-RBD protein production

The coding sequence for the RBD gene fused to a C-terminal 6 × His-tag was ligated into pFastBac vector. Cultures were performed in a 7 L bioreactor (Sartorius Biobraun, Sartorius, Germany), containing 2 L as a working volume, equipped with a pitch blade impeller. During the cell proliferation step, the following conditions were maintained: dissolved oxygen was regulated at 50% air saturation by continuous surface aeration. The temperature was maintained at 27 °C and the agitation rate at 120 rpm.

A batch culture was first started at 1 × 10⁶ cells/mL. When cell density reached 3 × 10⁶ cells/mL, S-f9 cells were infected with recombinant baculovirus at an MOI of 3. The expressed rRBD protein was collected 72 hours postinfection and then purified and tested by using human sera and anti-His antibody in Western blotting. The results demonstrate that the S-RBD protein has been perfectly expressed and purified as shown in Fig. 1B and 1C.

3.2. Development and validation of N- and S-RBD-based ELISA in Tunisia

ELISA assays for IgG antibodies to the S-RBD and N proteins were subsequently developed and optimized. The performance of both ELISA tests was assessed using a panel of sera from 108 patients with RT-PCR confirmed COVID-19 (collected within the first 6 weeks after the onset of illness symptoms) and 72 prepandemic sera collected prior to 2018 and cryopreserved within our biobank. As shown in Fig. 2 A and 2D, COVID-19 sera exhibited significantly higher ODs than prepandemic control sera in both tests (P < 0.0001). In addition, IgG antibodies' reactivity to N and S-RBD proteins was significantly higher in sera collected after days 8 and 15, respectively, compared to those collected earlier (Fig. 2B and 2E). The highest levels were obtained with sera collected between day 15 and day 28. The performance of both tests was estimated by the area under the curve (AUC) established using the RT-PCR test as a gold standard. The overall performance of the anti-N and anti-S-RBD ELISA was very high (AUC 0.966 and 0.98, respectively, p < 0.0001) with a sensitivity of 94% and a specificity of 93% for the anti-N test and a sensitivity of 95% and a specificity of 93% for the anti-S-RBD ELISA assay (Fig. 2C and 2F). Interestingly, levels of IgG anti-N and IgG anti-S-RBD were positively correlated in all tested patients (Rho = 0.66, P < 0.0001, Fig. S1).

Fig. 2.

N- and S-RBD-based ELISA assays validation. A total of 108 sera/plasma samples obtained from COVID-19 patients were confirmed by PCR and 72 sera samples collected before the pandemic (prior to 2018) from prepandemic sera were subjected to antibody detection. (A, D) N- and S-RBD specific IgG levels are presented as optical densities (OD450nm–620nm); (B, E) N and S-RBD specific IgG levels obtained in COVID-19 patients were stratified according to the blood collection time after the onset of the symptoms; (C, F) ROC curve analysis representing the performance of the N- and S-RBD based ELISA tests performances were estimated by the area under the ROC curve (AUC) established using the RT-PCR test as a gold standard. Bars indicate median values. Mann-Whitney t-test was used to compare differences between the 2 groups, a 2-tailed P-value <0.05 was considered statistically significant. *** P ≤ 0.001, ** P ≤ 0.01 and *P ≤ 0.05.

To better evaluate the performances of our ELISA based on N and S-RBD proteins, we did a head-to-head comparison on a set of negative (n = 42) and positive cases (n = 46) of our tests with the automated Roche Diagnostics. Indeed, it has been demonstrated that both Roche N and Roche S-RBD assays displayed the highest performance of automated SARS-CoV-2 serological immunoassays [15]. The results of all tests were read blindly to each other. Our assays demonstrated 95%/93% positive agreement and 93%/98% negative percent agreement with Roche Diagnostics for the N-based and S-RBD-based ELISA, respectively (Cohen's kappa value of 0.88 and 0.9 for N and S-RBD, respectively) ( Table 2).

Table 2.

Head-to-head comparison of the developed in-house ELISA assays with the commercial kits in different settings.

S-RBD-based ELISA
	Commercial kits		Positive	Negative	Total	Kappa value	Agreementa
Tunisia	Roche RBD assay	Positive	42	3	45	0.909	Almost perfect agreement
		Negative	1	42	43
		Total	43	45	88
Madagascar	WANTAI	Positive	55	12	67	0.672	Substantial agreement
		Negative	1	22	23
		Total	56	34	90
Morocco	EUROIMMUN assay	Positive	46	1	47	0.868	Almost perfect agreement
		Negative	2	19	21
		Total	48	20	68

N-based ELISA
	Commercial kits		Positive	Negative	Total	Kappa value	Agreementa
Tunisia	Roche N assay	Positive	42	2	44	0.886	Almost perfect agreement
		Negative	3	41	44
		Total	45	43	88
Madagascar	IDvet	Positive	60	1	61	0.664	Substantial agreement
		Negative	11	18	29
		Total	71	19	90
Senegal	IDvet	Positive	62	1	63	0.780	Substantial agreement
		Negative	9	29	38
		Total	71	30	101

^aKappa <0: No agreement; 0.00< Kappa <0.20: Slight agreement; 0.21< Kappa <0.40: Fair agreement; 0.41< Kappa <0.60: Moderate agreement; 0.61< Kappa <0.80: Substantial agreement; 0.81< Kappa <1.00: Almost perfect agreement.

3.3. Validation of the in-house ELISA tests in other African countries

To assess the performance of the developed ELISA in the context of African settings characterized by different backgrounds to endemic infections, additional cohorts of individuals living in Dakar (Senegal), and Casablanca (Morocco) were included. The overall performance of the anti-N and anti-S-RBD ELISA was high (AUC >0.97 and >0.94, respectivelyP < 0.0001) with a sensitivity from 91% to 98% and a specificity from 90% to 95% for the anti-S-RBD test and a sensitivity of 92% and specificity from 93% to 94% for the anti-N test (Table 1).

Table 1.

Anti N- and S-RBD-based ELISA assay performances in different settings.

S-RBD-based ELISA test
	Prepandemic sera (n)	COVID-19 patients (n)	AUC (95%CI)	P value	Sensitivity (95%CI)	Specificity (95%CI)
Senegal	106	67	0.9428 (0.9081– 0.9775)	<0.0001	91.04 (81.81– 95.83)	90.57 (83.50– 94.79)
Morocco	91	91	0.9942 (0.9882– 1.0000)	<0.0001	98.9 (94.04– 99.94)	95.6 (89.24– 98.28)

N-based ELISA test
	Prepandemic sera (n)	COVID-19 patients (n)	AUC (95%CI)	P value	Sensitivity (95%CI)	Specificity (95%CI)
Senegal	106	67	0.9701 (0.9447–0.9956)	<0.0001	92.54 (83.69–96.77)	94.34 (88.20–97.38)
Morocco	113	112	0.9735 (0.9569– 0.9900)	<0.0001	91.96 (85.29– 96.26)	93.81 (87.65– 97.47)

AUC = Area under the curve; CI = confidence interval.

The prepandemic control sera from Senegal were obtained from individuals who had various infections and/or pathologic conditions. Hence, we compared the reactivity of the developed assays according to these specific conditions. As shown in Fig. 3 A and 3D, a significant reactivity against N and S-RBD antigens were detected in sera from patients who tested seropositive for Chikungunya or malaria or from patients exhibiting rheumatoid factors. As malaria is endemic throughout Senegal, all included prepandemic samples were subjected to additional serological testing for IgG antibodies against Plasmodium falciparum antigens. As shown in Fig. 3B and 3E, the reactivity against either N or S-RBD antigens was significantly higher in seropositive patients for malaria than negative ones (P < 0.0001). The ELISA performances were then reanalyzed considering only subjects tested seronegative for malaria. In this specific group, we got a clear improvement in test performances with an AUC of 0.984, a sensitivity of 92%, and a specificity of 97% for anti-N test and an AUC of 0.988, a sensitivity of 97% and a specificity of 97% for the anti-S-RBD test (Fig. 3C and 3F).

Fig. 3.

Reactivity against N (A) and S-RBD (D) antigens in control patients negative for SARS-CoV-2 but suffering from various infections or pathologies: viral diseases including infection with human immunodeficiency virus (HIV), Influenza A/B (Flu), Chikungunya (CHIKV), Yellow fever (YFV), patients with rheumatoid factors (RF) and patients with positive or negative malaria serology. IgG anti-N (B) and anti-S-RBD (E) antibodies were compared in patients with positive (n = 72) or negative (n = 34) serology for malaria. ROC curves were established for the N-based (C) and S-RBD-based (F) ELISA tests using the sera from patients with negative serology for malaria as negative controls. Mann-Whitney t-test was used to compare differences between both groups, a 2-tailed P-value <0.05 was statistically significant. ****P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01 and *P ≤ 0.05.

Finally, we conducted in Senegal, Morocco and, also in Madagascar, a head-to-head comparison of our tests with 3 commercial kits, namely the WANTAI SARS-CoV-2 IgG ELISA and EUROIMMUN assay (for antibodies to S protein) and IDvet kit (for antibodies to the N protein). The comparison demonstrated good-to very good agreement between the tests (Table 2 ).

4. Discussion

During SARS-CoV-2 infection, the N, the S and the receptor binding domain of the spike antigen (S-RBD) are the main immunodominant antigens [[16], [17]] thus constituting good targets for serological tests used in seroprevalence studies. The latter is essential to assess the true diffusion of SARS-CoV-2 in exposed populations and detect asymptomatic infections that would otherwise escape detection. On another hand, S-RBD and N-based serological assays could also inform on the duration of antibodies generated by different vaccines. To this end, several immunoenzymatic techniques have been developed, optimized, and validated.

ELISA that detects antibodies to SARS-CoV-2 exhibited similar performance to the S-Flow assay and the luciferase system (LIPS) assay that recognized diverse SARS-CoV-2 antigens in immunoprecipitation [10]. In the present study, we developed and optimized indirect ELISA assays to detect SARS-CoV-2 specific IgG antibodies in serum samples using 2 recombinant forms of the viral proteins N and S-RBD that we successfully produced and purified in large amounts.

Our in-house developed ELISA tests based on S-RBD or N recombinant proteins were able to efficiently discriminate between sera from patients with COVID-19 and those collected in the prepandemic era as assessed by the ROC curve analysis. Indeed, when using a cut-off value of 0.52, the N-based ELISA assay had a sensitivity of 95% (95% CI: 93%–100%) and a specificity of 93% (95% CI: 97%-100%). The N-based developed assay has better performances than certain in-house as well as commercial tests based on the same antigen. Accordingly, Tehrani et al. [7] demonstrated that the EDI nucleocapsid IgG ELISA test showed 84%/95% of sensitivity and specificity, respectively. In addition, their in-house N-based ELISA IgG demonstrated a lower sensitivity (89%) but higher specificity (98%) than ours suggesting that some cross-reactive antibodies are detected in Tunisian prepandemic sera as compared to the US cohort used in their study. The RBD of the SARS-CoV-2 Spike protein is a very specific target for anti-SARS-CoV-2 antibody detection. Neutralizing antibodies are mapped to this domain, linking antibody response to RBD with neutralizing potential [18]. Our in-house S-RBD IgG developed assay gives 95%/93% of sensitivity and specificity, respectively when using a cut-off value of 0.27. Mehdi et al. [15] developed an in-house RBD-based IgG ELISA with higher specificity than our assay (99% vs 93%) but lower sensitivity. Indeed, the sensitivity found in the 3 sera panels they tested based on the onset of symptoms was less than 88%. Similarly, the in-house trimer spike IgG developed by Tehrani et al. [7] showed similar performances with 90%/99% of sensitivity and specificity, respectively. Both studies demonstrated that their assays had higher performances than the commercial ELISA EUROIMMUN assay, using spike S1 domain containing the RBD, suggesting that our S-RBD IgG test could have better performances than commercial assays mainly in terms of sensitivity. Our data also showed a relatively good correlation between N and S-RBD antibodies in COVID-19 patients in the first 6 weeks post-infection with r =0.66, a result corroborated with studies by different groups [[10], [19]].

Interestingly, the good performance of our tests has been also confirmed in other settings of African countries namely Morocco and Senegal. The overall performance of the anti-N and anti-S-RBD ELISA assays was high (AUC> 0.97 and >0.94, respectively, P < 0.0001) with a sensitivity varying from 91% to 98% and a specificity varying from 90% to 95% for both anti-N and -S-RBD tests. However, some African settings are characterized by a high prevalence of endemic infections such as malaria, with the dominance of the Plasmodium falciparum species [20]. Several studies found high rates of false positivity for commercial SARS-CoV-2 serological assays in different regions due to cross-reactivity with endemic infections [21]. Both plasmodium and dengue virus were listed to be tested for cross-reactivity of COVID-19 serological assays [[22], [23]]. Such cross-reactivity could explain lower specificities of serological tests as has already been reported by Steinhardt et al. [24] using prepandemic samples from Nigeria reported a lower specificity of Abbott Architect IgG and EUROIMMUN NCP IgG assays than the 1 claimed by the manufacturer. Using prepandemic samples from Senegal where malaria is endemic, our results also show significant reactivity against either N or S-RBD antigens in samples tested seropositive for malaria. Accordingly, the exclusion of malaria antibody positive sera from analysis clearly improves the performances of S-RBD and N-based tests. In 2 retrospective studies, an overall cross-reactivity of 4%-20% was found with prepandemic sera from malaria infected patients [[25], [26]]. Cross-reactivity of Plasmodium falciparum has been reported with either S or N SARS-CoV-2 proteins. One study showed that acute malaria infection induces antibody reactivity to the S1 Spike protein through recognition of the sialic acid moiety on N-linked glycans [27]. Another study reported that Plasmodium falciparum shared immunodominant epitopes with N and open reading frame 1ab of SARS-CoV-2 that could account for cross-reactivity [28]. Despite such cross-reactivities, the performances of our developed ELISA assays remain good with a strong agreement with various commercial tests, particularly with the Roche S-RBD assay, is, recognized as having the highest performances among automated serological immunoassays of SARS-CoV-2. Taking this into account, we do recommend that any potential user, of developed assays, from malaria endemic setting, should first set up the tests using malaria free control sera. It would also be noteworthy, to estimate the proportion of cross-reacting sera before starting large sero-epidemiological studies and add the necessary adjustments to the frequencies obtained.

Obviously, our study has some limitations. The first one is that the sampling was not performed simultaneously, thus SARS-CoV-2 variants infecting patients were not identified in each site. Despite this, the performances of the developed tests were quite comparable between the different sites. Moreover, developed assays were head-to-head compared with variable commercial assays available in each site. Yet, the comparisons demonstrated good-to very good agreement between the tests. Another limitation was the semi-qualitative feature of the assays. However, and thanks to the availability of WHO standardized sera, our assays can be made quantitative.

5. Conclusions

Herein, we report the development of in-house anti-N and anti-S-RBD ELISA assays that exhibited excellent performances across various endemic settings in Africa. Interestingly, developed ELISA assays were estimated to be at least 5 times cheaper per reaction/test than those commercial assays thus supporting their usefulness in low- and middle-income countries. The assays were developed in the frame of a collaborative project on the COVID-19 pandemic led by a consortium of 10 African countries and are currently used for seroprevalence studies on COVID-19 [29] in African populations as well as to understand the kinetics of humoral immune response induced by vaccines against COVID-19.