key: cord-0756855-fjsuo3yy
authors: Hoste, Alexis C.R.; Venteo, Angel; Fresco-Taboada, Alba; Tapia, Istar; Monedero, Alejandro; López, Lissette; Jebbink, Maarten F.; Pérez-Ramírez, Elisa; Jimenez-Clavero, Miguel Angel; Almonacid, Mercedes; Muñoz, Patricia; Guinea, Jesus; Vela, Carmen; van der Hoek, Lia; Rueda, Paloma; Sastre, Patricia
title: Two serological approaches for detection of antibodies to SARS-CoV-2 in different scenarios: A screening tool and a point-of-care test
date: 2020-08-11
journal: Diagn Microbiol Infect Dis
DOI: 10.1016/j.diagmicrobio.2020.115167
sha: c0c7c4d443e26d057af219d4b952d1602245e670
doc_id: 756855
cord_uid: fjsuo3yy

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected more than 8 million people worldwide, becoming a pandemic. Detecting antibodies against SARS-CoV-2 is of utmost importance and a good indicator of exposure and circulation of the virus within the general population. Two serological tools based on a Double Recognition assay (Enzyme-Linked Immunosorbent Assay, DR-ELISA and Lateral Flow Assay, DR-LFA) to detect total antibodies to SARS-CoV-2, have been developed based on the recombinant nucleocapsid protein. A total of 1065 serum samples, including positive for COVID-19 and negative samples from healthy donors or infected with other respiratory pathogens, were analyzed. The results showed values of sensitivity between 91.2%–100%, and specificity of 100%–98.2%, for DR-LFA and DR-ELISA, respectively. No cross-reactivity against seasonal coronavirus (HCoV-NL63, HCoV-229E, HCoV-HKU1, HCoV-OC43) was found. These results demonstrate the importance of serology as a complementary tool to PCR, for follow up of recovered patients and identification of asymptomatic individuals.

In December 2019, a novel coronavirus of animal origin (the severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) emerged in the city of Wuhan, in China, with the ability for human-to-human transmission (1) . The associated disease, now named COVID- 19 , spread rapidly all over the world, and was declared a pandemic by the World Health Organization (WHO) on March 11th, 2020. Infection due to SARS-CoV-2 induces high rates of morbidity and mortality as described by the WHO (2) . A significant concern is how rapidly the virus spreads, due in major part to the high number of asymptomatically infected individuals which could be an important source of viral dissemination (3, 4, 5) .

Since, there is currently no vaccine available, neither efficient treatment, hygiene measures together with keeping social distance, are the main preventive ways to avoid the spread of the virus. Serological studies can be used to collect epidemiological information on the prevalence of SARS-CoV-2. Moreover, in cases of COVID-19 not detected by RT-PCR, the serological assays should be considered as a supplementary diagnostic tool, especially from the second week of illness, when the sensitivity of the current molecular tests decreases (6, 7) . Therefore, the aim of the present work was the development of serological tools to determine the presence of antibodies against SARS-CoV-2 in the population, as an indicator of an ongoing or previous infection.

As for many other coronaviruses, one of the main structural proteins of SARS-CoV-2 is the nucleocapsid (N) protein. The N protein shows high immunogenic activity and is abundantly expressed during infection (8, 9, 10) . These features make the N protein a potential target for serodiagnosis of SARS-CoV-2 infection. To date, some diagnostic methods have been developed based on the N protein, although validated methods are still J o u r n a l P r e -p r o o f Journal Pre-proof lacking to better understand the epidemiology of SARS-CoV-2. In the current study, a Double Recognition Enzyme-Linked Immunosorbent Assay (DR-ELISA) was developed to determine the presence of immunoglobulins of different classes (IgG, IgM and IgA) to SARS-CoV-2 in human serum, to support the diagnosis of COVID-19. In parallel with this screening tool, a point-of-care test, based also on a double recognition format (a Double Recognition Lateral Flow Assay, DR-LFA), and using the N protein as the target antigen, was produced to be used immediately and on site when there is suspicion for infection. A double recognition assay is based on the use of the same protein (in this case the N protein) as the target antigen and detection molecule, using the principle that antibodies possess multiple antigen binding regions (two for IgG, four for IgA and ten for IgM), allowing their binding to both the target and detection antigen. Double recognition tests have the advantage that they screen for all SARS-CoV-2 antibodies, regardless if it is IgA, IgG or IgM.

To carry out this study, a total of 1065 samples were analyzed with 380 samples from positive patients to COVID-19 and 685 negative samples collected before 2019 or from patients negative to COVID-19. Finally, a cohort of samples from patients infected with common-cold-coronavirus or respiratory pathogens that could potentially cross-react with SARS-CoV-2 were included in the study.

The results shown in this paper reinforce the potential utility of serological testing as a 

The The SARS-CoV-2 N protein was labeled with peroxidase according to the method described by Nakane and Kawaoi (13) , to be used as detector molecule in the DR-ELISA described below.

A Double Recognition ELISA was developed as previously described (14) at 1 mg/mL was used. Both reagents were dispensed in two parallel lines on nitrocellulose membrane (HF120, Merck Millipore). After drying for 5 min at 45°C, the membranes were sealed and stored at room temperature.

Black latex beads (Merck Millipore) were activated with EDC (1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N-Hydroxysuccinimide) and

then coupled to N protein at a surface concentration of 1 mg/m 2 and blue latex beads were conjugated with the control protein.

To prepare the conjugate solution, the N-latex and control-latex particles were diluted at a concentration of 0.15% each, in a 25 mM Tris-HCl pH 9.5 buffer. The mixture was dispensed onto the conjugate pad, dried for 30 min at 45°C and stored at room temperature.

To assemble the 30 cm master card, nitrocellulose membrane, conjugate pad, sample pad (Cytosep 1662, Ahlstrom-Munksjö) and wicking pad were pasted on a plastic backing with adhesive and covered with a protector film. The master card was then cut into strips of 4.2 mm width.

The test was designed to be used with serum, plasma and blood samples.

Journal Pre-proof Twenty microliters of blood or ten microliters of serum/plasma were applied to the sample pad followed by 110 μL of running buffer (Tris-HCl pH 7.5, NaCl, casein and NaN 3 ).

Results were interpreted 10 minutes after running buffer addition. A scale of the intensity of the signal of the test line from 1 to 10 was used, in order to give a semi-quantitative value for statistical purposes.

Data were statistically analyzed by a receiver-operator characteristics (ROC) curve analysis using the MedCalc® 10 software to establish the optimal cut-off value for each assay. Prior to the analyses, the samples were classified into positive or negative by PCR or by other commercial serological assays. Using the same software, Fisher's exact test was performed to determine the statistical dependence between the two assays developed.

The complete SARS-CoV-2 N protein was cloned in the pDEST17 vector, expressed in E.

coli and further purified by immobilized metal affinity chromatography. The highly purified N protein was analyzed by gel electrophoresis followed by Coomassie staining. A band of the expected molecular mass of the N protein (around 45 kDa) was observed ( Figure 1 ).

Journal Pre-proof Table 2 . Table 2 : Comparison between the DR-LFA and the DR-ELISA

To fully validate the DR-ELISA and DR-LFA, the potential cross-reactivity by antibodies Table 3 ). Table 3 : Cross-reactivity with other respiratory pathogens. Abbreviation: ND: not determined.

For the new emerging virus SARS-CoV-2, the routinely used technique for testing patients is the RT-PCR, which detects the RNA of the virus at early stages of the infection (6) . Fully validated serological tests are still missing as many of the commercial serological tests J o u r n a l P r e -p r o o f currently available for SARS-CoV-2 are poorly validated or display low sensitivity or specificity (15) . In order to determine the prevalence of antibodies in the population and to complement the nucleic acid detection assays, especially at later days after the onset of the symptoms, serological assays are required (8) . Detection of antibodies is the most valuable indicator of the immune status of a person, identifying patients that have had COVID-19

infection, and providing more accurate data related to risk of infection.

In the present study, we developed two serological assays using the recombinant N protein Table 1, a group of 14 serum samples from early days post infection, positive to COVID-19 by respiratory-PCR yet still negative in the commercial serological assay (with seroconversion a few days later) were also tested in our assays. Four of these sera were positive in the DR-ELISA and three in the DR-LFA, indicating that the DR-assays we developed are highly sensitive.

Regarding the specificity of the newly developed assays, we only found one sample positive to Mycoplasma pneumonia that gave a positive signal in the DR-ELISA.

Interestingly, no cross-reactivity by antibodies directed to seasonal Alpha-or

Betacoronavirus was observed in our DR-assays, in contrast with regular SARS-CoV-2 antibody tests that do sometimes detect antibodies induced by HCoV-OC43 infection (19) .

We tested samples from 452 individuals that were negative for the virus in respiratory material (PCR) and also negative in serological assays, yet they were collected in a high risk group (personal communications). Eleven serum samples showed a positive signal in the DR-ELISA. Five had a very high S/P (>30) and the others were found with lower S/P (between 6 and 10). Three of the positives with high S/P were tested also for confirmation purposes in the DR-LFA, and also in this test the samples showed positive signals. These results could indicate that our tests can give false positives, or that the patients had experienced a previous infection, yet this was not diagnosed by the commercial assays, maybe not fully validated so far. It could also be that the serum samples contained only IgA recognizing SARS-CoV-2, since commercial assays used for classification only detect IgM and IgG (20) .

The serological assays could be a great complementary tool to the nucleic acid detection assays, as in this study, out of 626 samples with a negative PCR in respiratory material, 174 serum samples could diagnose a SARS-CoV-2 infection via serology (see Table 1 ). In these J o u r n a l P r e -p r o o f 174 patients, 123 were positive only to IgG, 23 were positive only to IgM and 28 were positive to IgG and IgM. An 89% correspondence was found between the DR-ELISA and the commercial serological assay for the 151 samples positive to IgG or positive to IgG and

IgM, but only one of the IgM positive was found positive in the DR-ELISA. This could demonstrate the higher affinity of IgG compared to IgM (21) which could lead to lower specificity of serological assays specifically targeting IgM.

Double recognition assays are sensitive tests, yet they also offer two additional advantages.

First, it is a multi-specie test, detecting antibodies in human serum, but also in serum samples from other animal species, since it uses the target antigen as the detector molecule, instead of anti-specie antibody, and secondly, it detects total antibodies in a given sample.

Unlike the antibody response usually observed in other infectious diseases (first IgM followed by IgG), during COVID-19 infection, IgM and IgG antibody responses appear almost simultaneously (22, 23) . Similar results were described previously for SARS, where the IgM appeared at the same time as the IgA and IgG (8) . This shows the importance of having a test that detects total antibodies in serum.

Using the N protein of the SARS-CoV-2 as the coating antigen and as the enzymeconjugated antigen instead of enzyme-conjugated secondary antibody provides a specific and sensitive serological assay for detection of total antibodies to SARS-CoV-2. The two assays developed in this study have been fully validated and received the CE marking. 

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We thank Dr. Belén Rebollo for technical support at Eurofins Ingenasa and María García and Alejandro Soler for technical assistance at INIA-CISA. We also thank Dr. John N. Barr for critical reading of the manuscript and assistance with the English language.

Training Network (ITN) HONOURs under grant No 721367 (to Alexis C. R. Hoste).

The datasets generated and analyzed during this work are available from the corresponding author on reasonable request.