Simultaneous detection of antibody responses to multiple SARS-CoV-2 antigens by a Western blot serological assay.

The nucleic acid test is still the standard assessment for the diagnosis of coronavirus disease 2019 (COVID-19), which is caused by human infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to supporting the confirmation of disease cases, serological assays are used for the analysis of antibody status and epidemiological surveys. In this study, a single Western blot strip (WBS) coated with multiple Escherichia coli (E. coli)-expressed SARS-CoV-2 antigens was developed for comprehensive studies of antibody profiles in COVID-19 patient sera. The levels of specific antibodies directed to SARS-CoV-2 spike (S), S2, and nucleocapsid (N) proteins were gradually increased with the same tendency as the disease progressed after hospitalization. The signal readouts of S, S2, and N revealed by the multi-antigen-coated WBS (mWBS)-based serological assay (mWBS assay) also demonstrated a positive correlation with the SARS-CoV-2 neutralizing potency of the sera measured by the plaque reduction neutralization test (PRNT) assays. Surprisingly, the detection signals against the unstructured receptor-binding domain (RBD) purified from E. coli inclusion bodies were not observed, although the COVID-19 patient sera exhibited strong neutralizing potency in the PRNT assays, suggesting that the RBD-specific antibodies in patient sera mostly recognize the conformational epitopes. Furthermore, the mWBS assay identified a unique and major antigenic epitope at the residues 1148, 1149, 1152, 1155, and 1156 located within the 1127-1167 fragment of the S2 subunit, which was specifically recognized by the COVID-19 patient serum. The mWBS assay can be finished within 14-16 min by using the automatic platform of Western blotting by thin-film direct coating with suction (TDCS WB). Collectively, the mWBS assay can be applied for the analysis of antibody responses, prediction of the protective antibody status, and identification of the specific epitope. KEY POINTS: • A Western blot strip (WBS) coated with multiple SARS-CoV-2 antigens was developed for the serological assay. • The multi-antigen-coated WBS (mWBS) can be utilized for the simultaneous detection of antibody responses to multiple SARS-CoV-2 antigens. • The mWBS-based serological assay (mWBS assay) identified a unique epitope recognized by the COVID-19 patient serum.

Serology Assays Used in SARS-CoV-2 Seroprevalence Surveys Worldwide: A Systematic Review and Meta-Analysis of Assay Features, Testing Algorithms, and Performance.

Background: Many serological assays to detect SARS-CoV-2 antibodies were developed during the COVID-19 pandemic. Differences in the detection mechanism of SARS-CoV-2 serological assays limited the comparability of seroprevalence estimates for populations being tested. Methods: We conducted a systematic review and meta-analysis of serological assays used in SARS-CoV-2 population seroprevalence surveys, searching for published articles, preprints, institutional sources, and grey literature between 1 January 2020, and 19 November 2021. We described features of all identified assays and mapped performance metrics by the manufacturers, third-party head-to-head, and independent group evaluations. We compared the reported assay performance by evaluation source with a mixed-effect beta regression model. A simulation was run to quantify how biased assay performance affects population seroprevalence estimates with test adjustment. Results: Among 1807 included serosurveys, 192 distinctive commercial assays and 380 self-developed assays were identified. According to manufacturers, 28.6% of all commercial assays met WHO criteria for emergency use (sensitivity [Sn.] >= 90.0%, specificity [Sp.] >= 97.0%). However, manufacturers overstated the absolute values of Sn. of commercial assays by 1.0% [0.1, 1.4%] and 3.3% [2.7, 3.4%], and Sp. by 0.9% [0.9, 0.9%] and 0.2% [−0.1, 0.4%] compared to third-party and independent evaluations, respectively. Reported performance data was not sufficient to support a similar analysis for self-developed assays. Simulations indicate that inaccurate Sn. and Sp. can bias seroprevalence estimates adjusted for assay performance; the error level changes with the background seroprevalence. Conclusions: The Sn. and Sp. of the serological assay are not fixed properties, but varying features depending on the testing population. To achieve precise population estimates and to ensure the comparability of seroprevalence, serosurveys should select assays with high performance validated not only by their manufacturers and adjust seroprevalence estimates based on assured performance data. More investigation should be directed to consolidating the performance of self-developed assays.

Detection of SARS-CoV-2 antibodies in serum and dried blood spot samples of vaccinated individuals using a sensitive homogeneous proximity extension assay.

A homogeneous PCR-based assay for sensitive and specific detection of antibodies in serum or dried blood spots (DBS) is presented and the method is used to monitor individuals infected with or vaccinated against SARS-CoV-2. Detection probes were prepared by conjugating the recombinant spike protein subunit 1 (S1), containing the receptor binding domain (RBD) of SARS-CoV-2, to each of a pair of specific oligonucleotides. The same was done for the nucleocapsid protein (NP). Upon incubation with serum or DBS samples, the bi- or multivalency of the antibodies (IgG, IgA or IgM) brings pairs of viral proteins with their conjugated oligonucleotides in proximity, allowing the antibodies to be detected by a modified proximity extension assay (PEA). Anti-S1 and anti-NP antibodies could be detected simultaneously from one incubation reaction. This Antibody PEA (AbPEA) test uses only 1 µl of neat or up to 100,000-fold diluted serum or one ø1.2 mm disc cut from a DBS. All 100 investigated sera and 21 DBS collected prior to the COVID-19 outbreak were negative, demonstrating a 100% specificity. The area under the curve, as evaluated by Receiver Operating Characteristic (ROC) analysis reached 0.998 (95%CI: 0.993-1) for samples taken from 11 days after symptoms onset. The kinetics of antibody responses were monitored after a first and second vaccination using serially collected DBS from 14 individuals. AbPEA offers highly specific and sensitive solution-phase antibody detection without requirement for secondary antibodies, no elution step when using DBS sample in a simple procedure that lends itself to multiplex survey of antibody responses.

Impact of COVID-19 Infection, Vaccination, and Serological Response in Immune Thrombocytopenic Purpura Patients: A Single-Center Global Analysis.

Both SARS-CoV-2 infection and vaccination have raised concern in immune-mediated diseases, including immune thrombocytopenic purpura (ITP) considering risk of de novo ITP development and ITP recurrence. Here, we report on data from a single-center retrospective-prospective collection aiming to evaluate platelet (plt) dynamics in patients (pts) with chronic ITP after COVID-19 infection (before and after vaccination) and after the first, second and third vaccine doses. Furthermore, we analyzed the serological response after the first two doses of COVID-19 vaccination. A total of 64 pts currently followed for chronic ITP who experienced COVD-19 infection and/or vaccination with an available plt count before and after such events were included in the analysis. A low incidence of ITP exacerbation following vaccine sessions (6-16%) was observed in comparison with a high frequency of exacerbation and rescue treatment necessity after COVID-19 infection in unvaccinated pts (83%). Moreover, the lower ITP exacerbation rate observed in infected pts previously vaccinated (18%) suggests further protective effects in this population. Finally, a high seroconversion rate was observed, confirming data reported in previously published studies on immune cytopenia and rheumatological diseases, but more evidence is awaited to establish the clinical impact of serological response.

DEVELOPMENT AND VALIDATION OF AN ENZYME-LINKED IMMUNOASSAY KIT FOR DIAGNOSIS AND SURVEILLANCE OF COVID-19.

There is a massive demand to identify alternative methods to detect new cases of COVID-19 as well as to investigate the epidemiology of the disease. In many countries, importation of commercial kits poses a significant impact on their testing capacity and increases the costs for the public health system. We have developed an ELISA to detect IgG antibodies against SARS-CoV-2 using a recombinant viral nucleocapsid (rN) protein expressed in E. coli. Using a total of 894 clinical samples we showed that the rN-ELISA was able to detect IgG antibodies against SARS-CoV-2 with high sensitivity (97.5%) and specificity (96.3%) when compared to a commercial antibody test. After three external validation studies, we showed that the test accuracy was higher than 90%. The rN-ELISA IgG kit constitutes a convenient and specific method for the large-scale determination of SARS-CoV-2 antibodies in human sera with high reliability.

A HIGH-THROUGHPUT MULTIPLEXED MICROFLUIDIC DEVICE FOR COVID-19 SEROLOGY ASSAYS

The need to develop high-throughput diagnostic platforms for infectious diseases has never been more evident than with the emergence of SARS-CoV-2 and the ensued COVID-19 pandemic. Microfluidics, in tandem with its multiplexing capabilities, high sensitivity, and potential for automation, provides a unique advantage towards the development of high-throughput serological diagnostic platforms. Here, we present a microfluidic device that detects IgG or IgM raised against four SARS-CoV-2 antigens (spike, S;S1 subunit, S1;the receptor-binding domain, RBD;and nucleocapsid, N) from 50 serum samples in parallel. We validated the platform with a cross-sectional cohort of 66 samples from confirmed COVID-19 patients and a pre-pandemic control of 34 serum samples collected in 2018. The analysis of both antibodies against all four viral antigens provided a sensitivity of 90.4% and a specificity of 94.1%, with both parameters increasing to 100% in late-stage samples (21-30 days after symptoms onset). We expect our device to open the door to massive serological testing, impacting diagnostics, vaccine development, and epidemiological understanding of COVID-19. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

Development and validation of an enzyme-linked immunoassay kit for diagnosis and surveillance of COVID-19.

There is a massive demand to identify alternative methods to detect new cases of COVID-19 as well as to investigate the epidemiology of the disease. In many countries, importation of commercial kits poses a significant impact on their testing capacity and increases the costs for the public health system. We have developed an ELISA to detect IgG antibodies against SARS-CoV-2 using a recombinant viral nucleocapsid (rN) protein expressed in E. coli. Using a total of 894 clinical samples we showed that the rN-ELISA was able to detect IgG antibodies against SARS-CoV-2 with high sensitivity (97.5%) and specificity (96.3%) when compared to a commercial antibody test. After three external validation studies, we showed that the test accuracy was higher than 90%. The rN-ELISA IgG kit constitutes a convenient and specific method for the large-scale determination of SARS-CoV-2 antibodies in human sera with high reliability.

Performance evaluation of an automatic chemiluminescence immune platform for SARS-CoV-2 neutralizing antibody after vaccination in real world.

OBJECTIVE: Reliable high-throughput serological assays for SARS-CoV-2 antibodies present an important role in the strength and duration of immunity after vaccination. The study investigated the analytical and clinical performances of neutralizing antibodies (NTAb) assay by chemiluminescent (CLIA), and SARS-CoV-2 neutralizing antibody after vaccination in real world. METHODS: The analytical performances of CLIA for SARS-CoV-2 NTAb were evaluated, followed by the sensitivity and specificity identified with a PRNT test from 50 volunteers. Then, a cohort of vaccine recipients (n = 37) were tracked with SARS-CoV-2 NTAb assay at prior to vaccination, one, three and six months post two doses. In real world, a total of 737 cases were recruited from physical examination center in Shenzhen Luohu People's Hospital (from Jun to August 2021) to analyze vaccination status. RESULTS: Serological assays on the CLIA were found with excellent characteristics including imprecision, repeatability and linearity. Besides, it was robust to icterus, lipemia and hemolysis. The good sensitivity and specificity were obtained at 98% and 100%, respectively. NTAb results showed a high correlation with PRNT50 titers (r 0.61). Until July 2021, the BBIBP-CorV (76.3%) and Sinovac CoronaVac (20.5%) were the predominant vaccines injection in Shenzhen, China. Adolescent less than 18 years was the main unvaccinated group (52.1%). The seropositive rate of inactive SRAR-CoV-2 vaccines exceeded 97% after inoculation. The NTAb generated by Sinovac CoronaVac with the schedule of 0-56 days was found significantly lower than that by BBIBP-CorV (P < 0.001). The follow-up of NTAb changes in a cohort and the dynamic variation of NTAb in real world disclosed steep downward by almost three times for NTAb level occurred at three months post twice vaccinations. The seropositive ratio was at least 50% over 6 months. CONCLUSIONS: SARS-CoV-2 neutralizing antibodies assay show excellent analytical and clinical performances, and a high correlation with neutralizing activity. Anti-epidemic measures and the urgent trial of SARS-CoV-2 vaccine was calling for adolescents.

Comparison of SARS-CoV-2 Antibodies and Six Immunoassays in Pediatric and Adult Patients 12 Weeks After COVID-19.

Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific humoral immune persistence has been proposed to be affected by patients' characteristics. Moreover, available conflicting assay results are needed to be settled through comparative research with defined clinical specimens. Methods This prospective study investigated SARS-CoV-2-specific antibodies among 43 adults and 34 children at a mean of 12 weeks after the onset of COVID-19 symptoms using six serological assays and compared their performance. We used two Euroimmun (Euroimmun, Luebeck, Germany), two automated Roche Elecsys (Basel, Switzerland), and two rapid immuno-chromatographic Ecotest (Matrix Diagnostics, Assure Tech. (Hangzhou) Co., L, China) assays to investigate SARS-CoV-2 antibodies. Results The findings showed that the Roche Elecsys anti-S total test yielded the best positivity/sensitivity (children 94.1% and adults 93.0%; p = 0.877) while five immunoglobulin IgG targeting assays had similar positivity/sensitivity between children (88.2% to 94.1%) and adults (88.4% to 93.0%) (p > 0.05). Although IgM positivity was relatively low (p < 0.001), it was found in the majority of our pediatric and adult patients (67.6% and 86.0%, respectively; p = 0.098). SARS-CoV-2 S IgG titers were found to be higher among males in pediatric and adult groups compared to females (p = 0.027 and p = 0.041, respectively). Furthermore, we observed significantly higher antibody titers among pneumonia patients (p = 0.001). Conclusion Overall, we concluded SARS-CoV-2 antibody persistence over an average of 12 weeks after the onset of COVID-19 symptoms. While automated Roche Elecsys total antibody assays yielded the best sensitivity (> 90%) and five assays targeting IgG had acceptable performance. Patients with pneumonia and males have higher antibody titers. The effect of antibody persistence on re-infections should be monitored in longitudinal studies.

Comprehensive mapping of SARS-CoV-2 peptide epitopes for development of a highly sensitive serological test for total and neutralizing antibodies.

Quantification of the anti-SARS-CoV-2 antibody response has proven to be a prominent diagnostic tool during the COVID-19 pandemic. Antibody measurements have aided in the determination of humoral protection following infection or vaccination and will likely be essential for predicting the prevalence of population level immunity over the next several years. Despite widespread use, current tests remain limited in part, because antibody capture is accomplished through the use of complete spike and nucleocapsid proteins that contain significant regions of overlap with common circulating coronaviruses. To address this limitation, a unique epitope display platform utilizing monovalent display and protease-driven capture of peptide epitopes was used to select high affinity peptides. A single round of selection using this strategy with COVID-19 positive patient plasma samples revealed surprising differences and specific patterns in the antigenicity of SARS-CoV-2 proteins, especially the spike protein. Putative epitopes were assayed for specificity with convalescent and control samples, and the individual binding kinetics of peptides were also determined. A subset of prioritized peptides was used to develop an antibody diagnostic assay that showed low cross reactivity while detecting 37% more positive antibody cases than a gold standard FDA EUA test. Finally, a subset of peptides were compared with serum neutralization activity to establish a 2 peptide assay that strongly correlates with neutralization. Together, these data demonstrate a novel phage display method that is capable of comprehensively and rapidly mapping patient viral antibody responses and selecting high affinity public epitopes for the diagnosis of humoral immunity.

Antibody Responses to BNT162b2 Vaccination in Japan: Monitoring Vaccine Efficacy by Measuring IgG Antibodies against the Receptor-Binding Domain of SARS-CoV-2.

To fight severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), mass vaccination has begun in many countries. To investigate the usefulness of a serological assay to predict vaccine efficacy, we analyzed the levels of IgG, IgM, and IgA against the receptor-binding domain (RBD) of SARS-CoV-2 in the sera from BNT162b2 vaccinated individuals in Japan. This study included 219 individuals who received two doses of BNT162b2. The levels of IgG, IgM, and IgA against RBD were measured by enzyme-linked immunosorbent assay before and after the first and second vaccination, respectively. The relationship between antibody levels and several factors, including age, gender, and hypertension were analyzed. Virus-neutralizing activity in sera was measured to determine the correlation with the levels of antibodies. A chemiluminescent enzyme immunoassay (CLEIA) method to measure IgG against RBD was developed and validated for the clinical setting. The levels of all antibody isotypes were increased after vaccination. Among them, RBD-IgG was dramatically increased after the second vaccination. The IgG levels in females were significantly higher than in males. There was a negative correlation between age and IgG levels in males. The IgG levels significantly correlated with the neutralizing activity. The CLEIA assay measuring IgG against RBD showed a reliable performance and a high correlation with neutralizing activity. Monitoring of IgG against RBD is a powerful tool to predict the efficacy of SARS-CoV-2 vaccination and provides useful information in considering a personalized vaccination strategy for COVID-19. IMPORTANCE Mass vaccination campaigns using mRNA vaccines against SARS-CoV-2 have begun in many countries. Serological assays to detect antibody production may be a useful tool to monitor the efficacy of SARS-CoV-2 vaccination in individuals. Here, we reported the induction of antibody isotype responses after the first and second dose of the BNT162b2 vaccine in a well-defined cohort of employees in Japan. We also reported that age, gender, and hypertension are associated with differences in antibody response after vaccination. This study not only provides valuable information with respect to antibody responses after BNT162b2 vaccination in the Japanese population but also the usefulness of serological assays for monitoring vaccine efficacy in clinical laboratories to determine a personalized vaccination strategy for COVID-19.

Anti-SARS-CoV-2 Immunoglobulin Isotypes, and Neutralization Activity Against Viral Variants, According to BNT162b2-Vaccination and Infection History.

Purpose: To compare SARS-CoV-2 antigen-specific antibody production and plasma neutralizing capacity against B.1 wild-type-like strain, and Gamma/P.1 and Delta/B.1.617.2 variants-of-concern, in subjects with different Covid-19 disease and vaccination histories. Methods: Adult subjects were: 1) Unvaccinated/hospitalized for Covid-19; 2) Covid-19-recovered followed by one BNT162b2 vaccine dose; and 3) Covid-19-naïve/2-dose BNT162b2 vaccinated. Multiplex Luminex® immunoassays measured IgG, IgA, and IgM plasma levels against SARS-CoV-2 receptor-binding domain (RBD), spike-1 (S), and nucleocapsid proteins. Neutralizing activity was determined in Vero E6 cytopathic assays. Results: Maximum anti-RBD IgG levels were similar in Covid-19­recovered individuals 8‒10 days after single-dose vaccination and in Covid-19-naïve subjects 7 days after 2nd vaccine dosing; both groups had ≈2­fold higher anti-RBD IgG levels than Unvaccinated/Covid-19 subjects tracked through 2 weeks post-symptom onset. Anti-S IgG expression patterns were similar to RBD within each group, but with lower signal strengths. Viral antigen-specific IgA and IgM levels were more variable than IgG patterns. Anti-nucleocapsid immunoglobulins were not detected in Covid-19-naïve subjects. Neutralizing activity against the B.1 strain, and Gamma/P.1 and Delta/B.1.617.2 variants, was highest in Covid­19-recovered/single-dose vaccinated subjects; although neutralization against the Delta variant in this group was only 26% compared to B.1 neutralization, absolute anti-Delta titers suggested maintained protection. Neutralizing titers against the Gamma and Delta variants were 33‒77% and 26‒67%, respectively, versus neutralization against the B.1 strain (100%) in the three groups. Conclusion: These findings support SARS-CoV-2 mRNA vaccine usefulness regardless of Covid-19 history, and confirm remarkable protection provided by a single vaccine dose in people who have recovered from Covid-19.

Kinetics of SARS-CoV-2-Neutralising Antibodies of Residents of Long-Term Care Facilities.

INTRODUCTION: Elderly residents of nursing homes (NHs) and long-term care units (LTCUs) have been shown to have a high risk of mortality and morbidity in cases of SARS-CoV-2 infection. The objective of this study was to examine the kinetics of neutralizing antibodies (NAbs) directed against the SARS-CoV-2 virus in residents of the NH and LTCU units of our University Hospital who were identified with positive serology after the first epidemic outbreak. MATERIALS AND METHODS: The participants included were sampled every three months for qualitative serological testing, as well as quantitative testing by neutralization tests using retroviral particles containing the S glycoprotein of SARS-CoV-2. Vaccination using the Comirnaty (Pfizer BNT162b2) vaccine begun before the last serological follow-up. RESULTS: The median NAb titer in June 2020 was 80 [40; 60] versus 40 [40; 160] three months later, showing a statistically significant decline (p < 0.007), but remained stable between the three- and six-month timepoints (p = 0.867). By nine months after vaccination, we observed a significant difference between vaccinated residents known to have positive serology before vaccination (SERO+, Vacc+) and those vaccinated without having previously shown COVID-19 seroconversion (SERO-, Vacc+), the latter group showing similar titers to the SERO+, Vacc- participants (p=0.166). The median antibody titer in SERO+, Vacc+ patients increased 15-fold following vaccination. DISCUSSION: Humoral immunity against SARS-CoV-2 appears to be persistent in elderly institutionalized patients, with a good post-vaccination response by residents who had already shown seroconversion but a notably diminished response by those who were seronegative before vaccination. To evaluate immunity in its entirety and elaborate a sound vaccination strategy, the cellular immune response via T cells specific to SARS-CoV-2 merits analysis, as this response is susceptible to being affected by immunosenescence.

Humoral anti-SARS-CoV-2 immune response after two doses of Comirnaty vaccine in nursing home residents by previous infection status.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is associated with increased morbidity and mortality in older adults. Although the advent of the first vaccines has significantly reduced these rates, data on older adults in clinical trials are scarce. OBJECTIVES: We quantified and compared the humoral response in individuals with vs. without pre-existing seropositivity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in a cohort of 69 patients living in a nursing home and who had received the recommended two doses of the Comirnaty (Pfizer-BioNTech®) vaccine. RESULTS: All 69 patients (100%) tested positive for antibodies against SARS-CoV-2 at 2 months post-vaccination. Residents with a pre-vaccination infection had significantly higher titers of anti-spike 1 IgG than those with no prior infection (median [interquartile range]: 55,726 [14463-78852] vs. 1314 [272-1249] arbitrary units, respectively; p < 0.001). The same result was observed for neutralizing antibodies titers (704 [320-1280] vs. 47 [20-40] respectively; p < 0.001). Between the pre-vaccination and post-vaccination periods, for IgG and neutralizing antibodies, we observed a 49 and 8-fold increase respectively. In comparison to the wild-type Receptor Binding Domain (RBD), the binding capacity of these vaccine sera was significantly decreased on the B.1.351 and P.1 variants RBD but not decreased with respect to the B.1.1.7 RBD. Although all nursing home residents developed a humoral response following Comirnaty vaccine, its intensity appeared to depend on the pre-vaccination serological status. CONCLUSION: Our results raise the question of how many doses of vaccine should be administered in older and how long the protection will be effective.

SARS-CoV-2 Antibody Testing in Health Care Workers: A Comparison of the Clinical Performance of Three Commercially Available Antibody Assays.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies are an excellent indicator of past COVID-19 infection. As the COVID-19 pandemic progresses, retained sensitivity over time is an important quality in an antibody assay that is to be used for the purpose of population seroprevalence studies. We compared 5,788 health care worker (HCW) serum samples by using two serological assays (Abbott SARS-CoV-2 anti-nucleocapsid immunoglobulin G (IgG) and Roche anti-SARS-CoV-2 anti-nucleocapsid total antibody) and a subset of samples (all Abbott assay positive or grayzone, n = 485) on Wantai SARS-CoV-2 anti-spike antibody enzyme-linked immunosorbent assay (ELISA). For 367 samples from HCW with a previous PCR-confirmed SARS-CoV-2 infection, we correlated the timing of infection with assay results. Overall, seroprevalence was 4.2% on Abbott and 9.5% on Roche. Of those with previously confirmed infection, 41% (150/367) and 95% (348/367) tested positive on Abbott and Roche, respectively. At 21 weeks (150 days) after confirmed infection, positivity on Abbott started to decline. Roche positivity was retained for the entire study period (33 weeks). Factors associated (P ≤ 0.050) with Abbott seronegativity in those with previous PCR-confirmed infection included sex (odds ratio [OR], 0.30 male ; 95% confidence interval [CI], 0.15 to 0.60), symptom severity (OR 0.19 severe symptoms; 95% CI, 0.05 to 0.61), ethnicity (OR, 0.28 Asian ethnicity; 95% CI, 0.12 to 0.60), and time since PCR diagnosis (OR, 2.06 for infection 6 months previously; 95% CI, 1.01 to 4.30). Wantai detected all previously confirmed infections. In our population, Roche detected antibodies up to at least 7 months after natural infection with SARS-CoV-2. This finding indicates that the Roche total antibody assay is better suited than Abbott IgG assay to population-based studies. Wantai demonstrated high sensitivity, but sample selection was biased. The relationship between serological response and functional immunity to SARS-CoV-2 infection needs to be delineated. IMPORTANCE As the COVID-19 pandemic progresses, retained sensitivity over time is an important quality in an antibody assay that is to be used for the purpose of population seroprevalence studies. There is a relative paucity of published literature in this field to help guide public health specialists when planning seroprevalence studies. In this study, we compared results of 5,788 health care worker blood samples tested by using two assays (Roche and Elecsys, anti-nucleocapsid antibody) and by testing a subset on a third assay (Wantai enzyme-linked immunosorbent assay [ELISA] anti-spike antibody). We found significant differences in the performance of these assays, especially with distance in time from PCR-confirmed COVID-19 infection, and we feel these results may significantly impact the choice of assay for others conducting similar studies.

Evaluation of Neutralizing Antibodies Against Highly Pathogenic Coronaviruses: A Detailed Protocol for a Rapid Evaluation of Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudovirus-Based Assay.

Emerging highly pathogenic human coronaviruses (CoVs) represent a serious ongoing threat to the public health worldwide. The spike (S) proteins of CoVs are surface glycoproteins that facilitate viral entry into host cells via attachment to their respective cellular receptors. The S protein is believed to be a major immunogenic component of CoVs and a target for neutralizing antibodies (nAbs) and most candidate vaccines. Development of a safe and convenient assay is thus urgently needed to determine the prevalence of CoVs nAbs in the population, to study immune response in infected individuals, and to aid in vaccines and viral entry inhibitor evaluation. While live virus-based neutralization assays are used as gold standard serological methods to detect and measure nAbs, handling of highly pathogenic live CoVs requires strict bio-containment conditions in biosafety level-3 (BSL-3) laboratories. On the other hand, use of replication-incompetent pseudoviruses bearing CoVs S proteins could represent a safe and useful method to detect nAbs in serum samples under biosafety level-2 (BSL-2) conditions. Here, we describe a detailed protocol of a safe and convenient assay to generate vesicular stomatitis virus (VSV)-based pseudoviruses to evaluate and measure nAbs against highly pathogenic CoVs. The protocol covers methods to produce VSV pseudovirus bearing the S protein of the Middle East respiratory syndrome-CoV (MERS-CoV) and the severe acute respiratory syndrome-CoV-2 (SARS-CoV-2), pseudovirus titration, and pseudovirus neutralization assay. Such assay could be adapted by different laboratories and researchers working on highly pathogenic CoVs without the need to handle live viruses in the BSL-3 environment.

Development of a Portable, Ultra-Rapid and Ultra-Sensitive Cell-Based Biosensor for the Direct Detection of the SARS-CoV-2 S1 Spike Protein Antigen.

One of the key challenges of the recent COVID-19 pandemic is the ability to accurately estimate the number of infected individuals, particularly asymptomatic and/or early-stage patients. We herewith report the proof-of-concept development of a biosensor able to detect the SARS-CoV-2 S1 spike protein expressed on the surface of the virus. The biosensor is based on membrane-engineered mammalian cells bearing the human chimeric spike S1 antibody. We demonstrate that the attachment of the protein to the membrane-bound antibodies resulted in a selective and considerable change in the cellular bioelectric properties measured by means of a Bioelectric Recognition Assay. The novel biosensor provided results in an ultra-rapid manner (3 min), with a detection limit of 1 fg/mL and a semi-linear range of response between 10 fg and 1 µg/mL. In addition, no cross-reactivity was observed against the SARS-CoV-2 nucleocapsid protein. Furthermore, the biosensor was configured as a ready-to-use platform, including a portable read-out device operated via smartphone/tablet. In this way, we demonstrate that the novel biosensor can be potentially applied for the mass screening of SARS-CoV-2 surface antigens without prior sample processing, therefore offering a possible solution for the timely monitoring and eventual control of the global coronavirus pandemic.

COVID-19: clinical presentation and detection methods.

The unending outburst of COVID-19 has reinforced the necessity of SARS-CoV-2 identification approaches for the prevention of infection transmission and the proper care of severe and critical patients. As there is no cure, a prompt and reliable diagnosis of SARS-CoV2 is vital to counter the spread and to provide adequate care and treatment for the infection. Currently, RT-PCR is a gold standard detection method for the qualitative and quantitative detection of viral nucleic acids. Besides, enzyme-linked immunosorbent assay is also a primarily used method for qualitative estimation of viral load. However, almost all the detection methods have their pros and cons in terms of specificity, accuracy, sensitivity, cost, time consumption, the need for sophisticated laboratories, and the requirement of skilled technical experts to carry out the detection tests. Thus, it is suggested to integrate different techniques to enhance the detection efficiency and accurateness for SARS-CoV2. This review focuses on preliminary, pre-confirmatory, and confirmatory methods of detection such as imaging techniques (chest-X-ray and chest- computed tomography), nucleic acid detection methods, serological assay methods, and viral culture and identification methods that are currently being employed to detect the presence of SARS-CoV-2 infection along with recent detection method and applicability for COVID-19.

Systematic evaluation of SARS-CoV-2 antigens enables a highly specific and sensitive multiplex serological COVID-19 assay.

OBJECTIVE: The COVID-19 pandemic poses an immense need for accurate, sensitive and high-throughput clinical tests, and serological assays are needed for both overarching epidemiological studies and evaluating vaccines. Here, we present the development and validation of a high-throughput multiplex bead-based serological assay. METHODS: More than 100 representations of SARS-CoV-2 proteins were included for initial evaluation, including antigens produced in bacterial and mammalian hosts as well as synthetic peptides. The five best-performing antigens, three representing the spike glycoprotein and two representing the nucleocapsid protein, were further evaluated for detection of IgG antibodies in samples from 331 COVID-19 patients and convalescents, and in 2090 negative controls sampled before 2020. RESULTS: Three antigens were finally selected, represented by a soluble trimeric form and the S1-domain of the spike glycoprotein as well as by the C-terminal domain of the nucleocapsid. The sensitivity for these three antigens individually was found to be 99.7%, 99.1% and 99.7%, and the specificity was found to be 98.1%, 98.7% and 95.7%. The best assay performance was although achieved when utilising two antigens in combination, enabling a sensitivity of up to 99.7% combined with a specificity of 100%. Requiring any two of the three antigens resulted in a sensitivity of 99.7% and a specificity of 99.4%. CONCLUSION: These observations demonstrate that a serological test based on a combination of several SARS-CoV-2 antigens enables a highly specific and sensitive multiplex serological COVID-19 assay.