key: cord-0683152-n2q91yln
authors: Fresco-Taboada, Alba; García-Durán, Marga; Aira, Cristina; López, Lissett; Sastre, Patricia; van der Hoek, Lia; van Gils, Marit J.; Brouwer, Philip J.M.; Sanders, Rogier W.; Holzer, Barbara; Zimpernikc, Irene; López-Collazo, Eduardo; Muñoz, Patricia; Rueda, Paloma; Vela, Carmen
title: Diagnostic performance of two serological assays for the detection of SARS-CoV-2 specific antibodies: surveillance after vaccination.
date: 2022-01-26
journal: Diagn Microbiol Infect Dis
DOI: 10.1016/j.diagmicrobio.2022.115650
sha: 289e0b7b1f184b8f4f6aba8398fd8dcd3623bc11
doc_id: 683152
cord_uid: n2q91yln

Massive vaccination programs are being carried out to limit the SARS-CoV-2 pandemic that started in December 2019. Serological tests are of major importance as an indicator of circulation of the virus and to assess how vaccine-induced immunity progresses. An Enzyme-Linked Immunosorbent Assay (ELISA) and a Lateral Flow Assay (LFA) have been developed based on the SARS-CoV-2 recombinant Receptor Binding Domain (RBD) and the combination of Spike and Nucleoprotein, respectively. The validation with 1272 serum samples by comparison with INgezim® COVID 19 DR showed good diagnostic performance (sensitivity: 93.2%-97.2%; specificity: 98.3%-99.3%) for detection of previous contact with SARS-CoV-2. Moreover, according to our results, these assays can help in the serosurveillance during and after vaccination, by detecting the humoral immune response as soon as 15 days post-vaccination and identifying low-respondents. Hence, these tests could play a key role in the progression to a COVID-19 free world, helping to adjust future vaccination protocols.

Since December 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has infected more than 260 million people and produced around 5.2 million deaths around the world [1] . As a complementary tool to direct detection of the virus by molecular detection techniques, plenty serological tests have been described and commercialized, some detecting nucleocapsid, spike specific antibodies or both [2] [3] [4] [5] [6] [7] .

As of January 2022, the European Medicines Agency (EMA) had recommended 5 vaccines for authorization in the EU, all of them containing the gene sequence that codifies for the Spike protein (S) [8] . Furthermore, the majority of the vaccines that are currently in clinical trials elicit antibodies that target the S protein, including the receptor binding domain (RBD) [9] .

S is a transmembrane glycoprotein that forms homotrimers protruding from the viral surface. This protein is formed by two functional subunits: S1 subunit, that binds to the host cell receptor through the RBD, and S2 subunit that allows the fusion of the viral and cellular membranes [10] .

Entrance of SARS-CoV-2 in the cells is carried out through the binding of S to hACE2 (human Angiotensin Converting Enzyme 2), with comparable affinity to S of SARS-CoV, that may contribute to its efficient spread among humans [11, 12] .

Although several epitopes within the S protein have been described to elicit neutralizing antibodies [13, 14] , most of those produced upon natural infection target the RBD, blocking the binding of the virus to hACE2 [15] . The Nucleoprotein does not elicit neutralizing antibodies, however, good correlation between antibody response to this protein and neutralizing antibody titer has been described [16] .

Enzyme-Linked Immunosorbent Assay (ELISA) is a well-known lab technology, that allows the simultaneous analysis of high numbers of samples. In contrast, lateral flow assays (LFA) are one of the most widely used techniques for point-of-care testing and diagnosis due to its characteristics (user-friendly, low cost, rapid results, long-term stability over a wide range of climates) [17] .

Since vaccines are mainly based on the S protein, it is important to develop tests to complement serological status determination. For that purpose, here, the comparison of an ELISA that detects RBD-specific antibodies, and a dual rapid test (LFA) for the differential detection of S and Nspecific antibodies, is described. A collection of serum samples from naturally infected, vaccinated non-infected, vaccinated previously infected, and non-infected nor vaccinated people, has been analyzed to validate the two assays and determine their performance.

A total of 1272 human serum samples were evaluated by the two assays described in this article.

Serum samples were collected from selected volunteers (SARS-CoV-2 naturally infected, vaccinated non-infected, vaccinated previously infected, and non-infected nor vaccinated) from different hospitals and laboratories ( Table 1 ). The sera from the Amsterdam UMC were collected through the Amsterdam Cohort Studies on HIV infection and AIDS. Samples were characterized as positive or negative to previous infection by the commercial CE-certified ELISA INgezim ® COVID 19 DR (N-ELISA) [2] for the statistical evaluations. Data relative to PCR were considered when available. The seroneutralization assay (SNT) previously described [18] was used as reference to determine the presence of neutralizing antibodies. Reference sera from the World Health Organization (WHO) were used for determination of the Limit of detection (LoD) for the different assays [19] .

Recombinant expression of Nucleoprotein was carried out as described [2] .

Recombinant expression of Spike trimeric and RBD proteins was performed by transfection of suspension cultures of Human embryonic kidney 293-F (HEK293) cells. The codon-optimized DNA sequence encoding the RBD residues 331-524 (GeneBank accession number NC_045512) was ordered to Integrated DNA Technologies, amplified by PCR, and inserted into the plasmid pCMV6-AC-FC-S (Origene), generating the plasmid pCMV6-RBDmFc. A second plasmid, pCMV6-RBDHis, was generated by modifying the described RBD sequence by PCR adding a 6histidine sequence at the 3' region followed by a stop codon. The transfections were performed in a proportion of 1 µg plasmid/ml cell culture, yielding recombinant proteins expressed as a C-terminal fusion with a mouse Fc (RBDmFc) or with a 6His tag (RBDHis). The proteins were collected from the supernatant of HEK293 cell suspension cultures four days post-transfection, and subsequently purified by affinity chromatography to protein G or Ni 2+ for RBDmFc and RBDHis, respectively. [20] .

S protein was expressed fused to a 6-histidine tag as previously described [21] . The transfection procedure was the same as for RBD proteins except that the culture was collected six days post transfection.

A DR-ELISA was developed as previously described [2, 22] with some modifications. The abovedescribed proteins were tested: RBDHis rendered better specificity results and RBDmFc was more suitable for conjugation. Briefly, 1 ng/µl of the RBD protein (RBDHis) was used to coat 96-well plates and was incubated overnight at 4 °C in 50 mM carbonate buffer, pH 9.6. After washing the wells with PBS, 0.05% Tween 20 (PBST) using a manual washer, a blocking step was performed with StabilZyme® SELECT Stabilizer (SurModics, Inc.) for 1 h at room temperature (RT). The plate was incubated with serum samples diluted 1:5 in PBST with 2.5% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) for 30 min at RT. Duplicates of positive (pool of human SARS-CoV-2 positive sera) and negative (dilution buffer) controls were included in each plate. The wells were washed as described above and incubated with the HRP-conjugated RBDmFc protein for 30 min at RT. Finally, after a washing step as above mentioned, the plate was incubated for 15 min with the substrate (TMB-MAX, Neogen Corporation), and the reaction was stopped by the addition of 0.5 M sulfuric acid. The absorbance was measured at 450 nm using a SpectraMax M5 plate reader (Molecular Devices, LLC). Results were presented as S/P, defined as: (sample ODnegative control OD) / (positive control OD -negative control OD) × 10.

This double LFA comprises two parallel strips, one for the detection of N-specific antibodies (N-LFA) and another one for S-specific antibodies (S-LFA). The single assay, for the detection of Nspecific antibodies, has been already described [2] . Thus, the assay for the detection of S-specific antibodies is reported.

Recombinant S protein diluted at 1 mg/mL in 20 mM Tris-HCl buffer, pH 7.5 containing sucrose and sodium azide was used as the test line capture reagent. As control line capture reagent, a monoclonal antibody against the control protein was employed. These reagents were dispensed in two parallel lines on a nitrocellulose membrane and the resulting membranes were dried for 5 min at 45°C, sealed and stored at room temperature under dry conditions.

The S protein was covalently conjugated to red latex beads whereas blue latex particles were covalently conjugated with the control protein. Prior to protein conjugation, bare latex beads were washed and activated with EDC and NHS. Then, S protein was coupled at a surface concentration of 1 mg/m 2 and, after blocking the non-reactive functional groups, particles were diluted to a concentration of 1% in Tris-HCl 10 mM pH 8.2.

In order to prepare the conjugate solution, the S-latex and control-latex particles were diluted at a concentration of 0.2% and 0.15% respectively, and the mixture was dispensed onto the conjugate pad, dried for 30 min at 45°C and stored at room temperature under dry conditions.

The nitrocellulose membrane, conjugate pad, sample pad and wicking pad were pasted on a plastic card and protected with a cover tape. The master card was then cut into strips of 4.2 mm width, and both N and S -specific Ab strips were assembled into cassettes.

The double test was designed to be used with serum or blood samples. Serum samples were fresh, refrigerated at 2-8°C or frozen at -20°C. Blood samples were fresh or refrigerated up to 4 days at 2-8°C and collected with anticoagulant (EDTA, heparin or citrate).

To perform the test, twenty microliters of blood or ten microliters of serum were applied to the round window of each cassette and followed by addition of 3 drops of the shared running buffer (Tris-HCl pH 7.5, NaCl, casein and NaN 3 ). Results were interpreted after 10 minutes.

MedCalc® 10 software was used for statistical analysis. Receiver Operating Characteristics (ROC) curves analyses were performed to establish the optimal cut-off value for each assay, as well as sensitivity, specificity, and likelihood ratio (LR). Regarding samples from vaccinated subjects, results were presented in box-whisker plots according to time post-vaccination.

The three proteins expressed (RBDmFc, RBDHis and S [21] ) showed the expected apparent molecular weights considering their potential N-glycosylation pattern (3, 2, and 6 potential Nglycosylation sites, respectively). The mean expressions yields were between 10 and 20 mg/L. The purity degree was >99%, according to Coomassie staining of SDS-PAGE gels ( Figure 1 ).

First, the potential diagnostic application of the newly developed assays was evaluated with samples collected from naturally infected and non-infected volunteers. Analyzing signal intensity (S/P for ELISA and test-line intensity for LFA), best cut off values were established according to a ROC curve analysis ( Figure 2 ). Figure 2 and detailed in Table 2 , the new assays exhibited good diagnostic parameters and agreement with the commercial ELISA used as reference in the present study. Moreover, all the assays exhibited a high positive LR (+LR), >10, and a low negative LR (-LR), indicating that the performance of the assays is optimal for diagnosing past infection. Additionally, the tests' LoD was assessed with WHO reference sera ( Table 2) . 

In order to determine potential cross-reactivity in the reported assays, samples containing antibodies specific to other viruses were analyzed (VIH, Adenovirus, Zoster Herpes, Rubeola, Citomegalovirus, Epstein-Barr, Hep C, Hep B, Influenza A and B). No cross-reactivity was observed with these viruses, except for one sample, positive for Epstein-Barr antibodies that was positive for S-specific antibodies when analyzed by S-LFA, but negative for the other assays.

Furthermore, samples containing antibodies to other human Coronavirus (hCoV) were analyzed by S-LFA (n=40 [23] ) and RBD-ELISA (n=10), being all of them negative by both assays, indicating no cross-reaction with any other hCoV.

Several analytes that could cause interferences in the assays were also studied [24] . Samples with an increased content of Beta human chorionic gonadotropin (β-hCG), bilirubin, hemoglobin or lipids were analyzed, showing that samples with concentrations up to 1.9 ng/dl β-hCG, 48 mg/dl hemoglobin, 800 mg/dl triglycerides and 7 mg/dl bilirubin respectively, do not affect the test result. Both assays showed a sensitivity in vaccinated and previously infected individuals of 100%. In some samples, N-specific antibodies were still detectable, whereas in others the titer had considerably decreased and was not detectable by neither ELISA nor LFA. As expected after vaccination, and in contrast to anti-S antibodies, anti-N antibodies decreased with time ( Figure 3D ).

In order to study the ability of the RBD-ELISA and S-LFA to detect neutralizing antibodies, the surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike proteinprotein interaction (cPass™ SARS-CoV-2 Neutralization Antibody Detection Kit, GeneScript, cPass™) was used in parallel, focusing on the antibodies specific to S protein. To 

Antibody tests that detect the responses to SARS-CoV-2 RBD and trimeric Spike protein can help in tracking the effect of vaccination programs.

In this article, two serological assays for the surveillance of the antibody response to infection and vaccination have been described: an ELISA that detects RBD-specific antibodies and a Dual rapid test that detects S and N-specific antibodies. Due to the high prevalence the disease can have, the simultaneous detection of N and S-specific antibodies could be helpful for the determination of the immunological status pre-and post-vaccination.

Both tests have demonstrated high sensitivity and specificity, as well as +LR and -LR that make them optimal assays for the diagnosis of SARS-CoV-2 infection. Good agreement has been obtained with all the assays compared to the commercial ELISA INgezim ® COVID 19 DR used as reference, as well as between assays specific for the detection of antibodies to the same protein.

When comparing tests for the detection of specific antibodies to the S or RBD proteins with the commercial ELISA (specific for the detection of N-antibodies), slightly lower values of specificity were observed. However, most of the samples classified as false positive also gave a positive result with a commercial assay with the ability to detect antibodies to S protein. If those samples were considered positive, the specificity would be 100% for RBD-ELISA and 99.6% for S-LFA. This observation may indicate that some of the people tested, developed immunoglobulins against N protein but not against S protein (4.3% of infected-individuals' samples) and vice versa (12.1% of infected-individuals samples) during SARS-CoV-2 infection and/or that the level of antibodies against N protein decreased with time, as some published data are pointing out [25, 26] . In the lateral flow assay, if a positive result to either N or S was considered positive, the sensitivity of the assay would increase, justifying the use of a dual test as the one described (INgezim ® COVID 19 N/S DUAL CROM).

Currently, there is a massive vaccination campaign with the aim to achieve herd immunity, which highlights the mayor importance to assess the durability of the immunity. This study showed that in vaccinated people previously infected, S-specific antibody levels measured by both tests were remarkably high, as early as 7 days after the first vaccine dose, independently of their N-specific antibody levels, as already described [4, 27] . In fact, 15 days after first dose, all the vaccinated and previously infected people, showed high antibody titer with all the tested vaccines. That result indicates that vaccination would act as a booster, as described by Wise [28] , enabling vaccine supplies to be deployed effectively.

On the other hand, there is a gradual increase in S-antibody levels after first vaccine in non-infected people, but some of them remained negative even at early times after second dose. However, 15 days after completing vaccination schedule, all of them seroconverted except one, which was confirmed negative by an alternative assay. Although the current vaccines have demonstrated to be highly immunogenic, the possibility of low responders exists, and that is one of the reasons why serosurveillance is important. Furthermore, according to a small study in a nursing-home corresponding to 48 users and 32 employees, the immunological response was age-dependent, since 97% of the employees (average of 47 years-old) showed a positive result 15 days after second vaccination dose, in contrast to only 65% of the elderly older than 85 years old (data not shown).

Thus, the detection of hyporesponsiveness groups [29, 30] 

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Writing -Original Draft, Visualization, Lissett López: Investigation, Writing -Review & Editing, Patricia Sastre: Resources, Writing -Review & Editing, Lia van der Hoek: Conceptualization, Resources, Writing -Review & Editing

Positive to antibodies specific against HCV, viremic (6) and nonviremic (6)

Positive to antibodies specific against another human Coronavirus: OC43 (14)

Austrian Agency for Health and Food Safety

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.