Development of a high-sensitivity ELISA detecting IgG, IgA and IgM antibodies to the SARS-CoV-2 spike glycoprotein in serum and saliva.

Detecting antibody responses during and after SARS-CoV-2 infection is essential in determining the seroepidemiology of the virus and the potential role of antibody in disease. Scalable, sensitive and specific serological assays are essential to this process. The detection of antibody in hospitalized patients with severe disease has proven relatively straightforward; detecting responses in subjects with mild disease and asymptomatic infections has proven less reliable. We hypothesized that the suboptimal sensitivity of antibody assays and the compartmentalization of the antibody response may contribute to this effect. We systematically developed an ELISA, optimizing different antigens and amplification steps, in serum and saliva from non-hospitalized SARS-CoV-2-infected subjects. Using trimeric spike glycoprotein, rather than nucleocapsid, enabled detection of responses in individuals with low antibody responses. IgG1 and IgG3 predominate to both antigens, but more anti-spike IgG1 than IgG3 was detectable. All antigens were effective for detecting responses in hospitalized patients. Anti-spike IgG, IgA and IgM antibody responses were readily detectable in saliva from a minority of RT-PCR confirmed, non-hospitalized symptomatic individuals, and these were mostly subjects who had the highest levels of anti-spike serum antibodies. Therefore, detecting antibody responses in both saliva and serum can contribute to determining virus exposure and understanding immune responses after SARS-CoV-2 infection.

Comparison of SARS-CoV-2 Antibody Responses and Seroconversion in COVID-19 Patients Using Twelve Commercial Immunoassays.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody assays have high clinical utility in managing the pandemic. We compared antibody responses and seroconversion of coronavirus disease 2019 (COVID-19) patients using different immunoassays. METHODS: We evaluated 12 commercial immunoassays, including three automated chemiluminescent immunoassays (Abbott, Roche, and Siemens), three enzyme immunoassays (Bio-Rad, Euroimmun, and Vircell), five lateral flow immunoassays (Boditech Med, SD biosensor, PCL, Sugentech, and Rapigen), and one surrogate neutralizing antibody assay (GenScript) in sequential samples from 49 COVID-19 patients and 10 seroconversion panels. RESULTS: The positive percent agreement (PPA) of assays for a COVID-19 diagnosis ranged from 84.0% to 98.5% for all samples (>14 days after symptom onset), with IgM or IgA assays showing higher PPAs. Seroconversion responses varied across the assay type and disease severity. Assays targeting the spike or receptor-binding domain protein showed a tendency for early seroconversion detection and higher index values in patients with severe disease. Index values from SARS-CoV-2 binding antibody assays (three automated assays, one LFIA, and three EIAs) showed moderate to strong correlations with the neutralizing antibody percentage (r=0.517-0.874), and stronger correlations in patients with severe disease and in assays targeting spike protein. Agreement among the 12 assays was good (74.3%-96.4%) for detecting IgG or total antibodies. CONCLUSIONS: Positivity rates and seroconversion of SARS-CoV-2 antibodies vary depending on the assay kits, disease severity, and antigen target. This study contributes to a better understanding of antibody response in symptomatic COVID-19 patients using currently available assays.

Diagnostic performance of four SARS-CoV-2 antibody assays in patients with COVID-19 or with bacterial and non-SARS-CoV-2 viral respiratory infections.

SARS-CoV-2 antibody assays are used for epidemiological studies and for the assessment of vaccine responses in highly vulnerable patients. So far, data on cross-reactivity of SARS-CoV-2 antibody assays is limited. Here, we compared four enzyme-linked immunosorbent assays (ELISAs; Vircell SARS-CoV-2 IgM/IgA and IgG, Euroimmun SARS-CoV-2 IgA and IgG) for detection of anti-SARS-CoV-2 antibodies in 207 patients with COVID-19, 178 patients with serological evidence of different bacterial infections, 107 patients with confirmed viral respiratory disease, and 80 controls from the pre-COVID-19 era. In COVID-19 patients, the assays showed highest sensitivity in week 3 (Vircell-IgM/A and Euroimmun-IgA: 78.9% each) and after week 7 (Vircell-IgG: 97.9%; Euroimmun-IgG: 92.1%). The antibody indices were higher in patients with fatal disease. In general, IgM/IgA assays had only limited or no benefit over IgG assays. In patients with non-SARS-CoV-2 respiratory infections, IgG assays were more specific than IgM/IgA assays, and bacterial infections were associated with more false-positive results than viral infections. The specificities in bacterial and viral infections were 68.0 and 81.3% (Vircell-IgM/IgA), 84.8 and 96.3% (Euroimmun-IgA), 97.8 and 86.0% (Vircell-IgG), and 97.8 and 99.1% (Euroimmun-IgG), respectively. Sera from patients positive for antibodies against Mycoplasma pneumoniae, Chlamydia psittaci, and Legionella pneumophila yielded particularly high rates of unspecific false-positive results in the IgM/IgA assays, which was revealed by applying a highly specific flow-cytometric assay using HEK 293 T cells expressing the SARS-CoV-2 spike protein. Positive results obtained with anti-SARS-CoV-2 IgM/IgA ELISAs require careful interpretation, especially if there is evidence for prior bacterial respiratory infections.

SARS-CoV-2 elicits robust adaptive immune responses regardless of disease severity.

BACKGROUND: The SARS-CoV-2 pandemic currently prevails worldwide. To understand the immunological signature of SARS-CoV-2 infections and aid the search and evaluation of new treatment modalities and vaccines, comprehensive characterization of adaptive immune responses towards SARS-CoV-2 is needed. METHODS: We included 203 recovered SARS-CoV-2 infected patients in Denmark between April 3rd and July 9th 2020, at least 14 days after COVID-19 symptom recovery. The participants had experienced a range of disease severities from asymptomatic to severe. We collected plasma, serum and PBMC's for analysis of SARS-CoV-2 specific antibody response by Meso Scale analysis including other coronavirus strains, ACE2 competition, IgA ELISA, pseudovirus neutralization capacity, and dextramer flow cytometry analysis of CD8+ T cells. The immunological outcomes were compared amongst severity groups within the cohort, and 10 pre-pandemic SARS-CoV-2 negative controls. FINDINGS: We report broad serological profiles within the cohort, detecting antibody binding to other human coronaviruses. 202(>99%) participants had SARS-CoV-2 specific antibodies, with SARS-CoV-2 neutralization and spike-ACE2 receptor interaction blocking observed in 193(95%) individuals. A significant positive correlation (r=0.7804) between spike-ACE2 blocking antibody titers and neutralization potency was observed. Further, SARS-CoV-2 specific CD8+ T-cell responses were clear and quantifiable in 95 of 106(90%) HLA-A2+ individuals. INTERPRETATION: The viral surface spike protein was identified as the dominant target for both neutralizing antibodies and CD8+ T-cell responses. Overall, the majority of patients had robust adaptive immune responses, regardless of their disease severity. FUNDING: This study was supported by the Danish Ministry for Research and Education (grant# 0238-00001B) and The Danish Innovation Fund (grant# 0208-00018B).

Comparison of the immune response following subcutaneous versus intranasal modified-live virus booster vaccination against bovine respiratory disease in pre-weaning beef calves that had received primary vaccination by the intranasal route.

This study was performed to elucidate whether the route of booster vaccination affects the immune response against respiratory vaccine viruses in pre-weaning beef calves that receive primary intranasal (IN) vaccination during the first month of life. The objective was to compare the serum neutralizing antibody (SNA) titers to BHV1, BRSV, and BPI3V, cytokine mRNA expression and mucosal BHV1- and BRSV-specific IgA in nasal secretions following administration of IN or subcutaneous (SC) modified-live virus (MLV) booster vaccines 60 days after primary IN vaccination in young beef calves. Twenty-one beef calves were administered 2 mL of an IN MLV vaccine containing BHV1, BRSV, and BPI3V (Inforce3®) between one and five weeks of age. Sixty days after primary vaccination, calves were randomly assigned to one of two groups: IN-MLV (n = 11): Calves received 2 mL of the same IN MLV vaccine used for primary vaccination and 2 mL of a SC MLV vaccine containing BVDV1 & 2 (Bovi- Shield GOLD® BVD). SC-MLV (n = 10): Calves were administered 2 mL of a MLV vaccine containing, BHV1, BRSV, BPI3V, and BVDV1 & 2 (Bovi-Shield GOLD® 5). Blood and nasal secretion samples were collected on days -61 (primary vaccination), -28, -14, 0 (booster vaccination), 14, 21, 28, 42 and 60 for determination of SNA titers, cytokine gene expression analysis and nasal virus-specific IgA concentrations. Statistical analysis was performed using a repeated measures analysis through PROC GLIMMIX of SAS®. Booster vaccination by neither IN nor SC routes induced a significant increase in SNA titers against BHV1, BRSV, and BPI3V. Subcutaneous booster vaccination induced significantly greater BRSV-specific SNA titers (on day 42) and IgA concentration in nasal secretions (on days 21 and 42) compared to calves receiving IN booster vaccination. Both IN and SC booster vaccination were able to stimulate the production of BHV1-specific IgA in nasal secretions. In summary, booster vaccination of young beef calves using either SC or IN route two months after IN MLV primary vaccination resulted in comparable SNA titers, cytokine gene expression profile and virus-specific IgA concentration in nasal secretions. Only a few differences in the systemic and mucosal immune response against BHV1 and BRSV were observed. Subcutaneous booster vaccination induced significantly greater BRSV-specific SNA and secretory IgA titers compared to IN booster vaccination.

Seroprevalence of SARS-CoV-2 Assessed by Four Chemiluminescence Immunoassays and One Immunocromatography Test for SARS-Cov-2.

The onset of the new SARS-CoV-2 coronavirus encouraged the development of new serologic tests that could be additional and complementary to real-time RT-PCR-based assays. In such a context, the study of performances of available tests is urgently needed, as their use has just been initiated for seroprevalence assessment. The aim of this study was to compare four chemiluminescence immunoassays and one immunochromatography test for SARS-Cov-2 antibodies for the evaluation of the degree of diffusion of SARS-CoV-2 infection in Salerno Province (Campania Region, Italy). A total of 3,185 specimens from citizens were tested for anti-SARS-CoV-2 antibodies as part of a screening program. Four automated immunoassays (Abbott and Liaison SARS-CoV-2 CLIA IgG and Roche and Siemens SARS-CoV-2 CLIA IgM/IgG/IgA assays) and one lateral flow immunoassay (LFIA Technogenetics IgG-IgM COVID-19) were used. Seroprevalence in the entire cohort was 2.41, 2.10, 1.82, and 1.85% according to the Liaison IgG, Abbott IgG, Siemens, and Roche total Ig tests, respectively. When we explored the agreement among the rapid tests and the serologic assays, we reported good agreement for Abbott, Siemens, and Roche (Cohen's Kappa coefficient 0.69, 0.67, and 0.67, respectively), whereas we found moderate agreement for Liaison (Cohen's kappa coefficient 0.58). Our study showed that Abbott and Liaison SARS-CoV-2 CLIA IgG, Roche and Siemens SARS-CoV-2 CLIA IgM/IgG/IgA assays, and LFIA Technogenetics IgG-IgM COVID-19 have good agreement in seroprevalence assessment. In addition, our findings indicate that the prevalence of IgG and total Ig antibodies against SARS-CoV-2 at the time of the study was as low as around 3%, likely explaining the amplitude of the current second wave.

Human Milk from Previously COVID-19-Infected Mothers: The Effect of Pasteurization on Specific Antibodies and Neutralization Capacity.

BACKGROUND: Since the outbreak of coronavirus disease 2019 (COVID-19), many put their hopes in the rapid availability of effective immunizations. Human milk, containing antibodies against syndrome coronavirus 2 (SARS-CoV-2), may serve as means of protection through passive immunization. We aimed to determine the presence and pseudovirus neutralization capacity of SARS-CoV-2 specific IgA in human milk of mothers who recovered from COVID-19, and the effect of pasteurization on these antibodies. METHODS: This prospective case control study included lactating mothers, recovered from (suspected) COVID-19 and healthy controls. Human milk and serum samples were collected. To assess the presence of SARS-CoV-2 antibodies we used multiple complementary assays, namely ELISA with the SARS-CoV-2 spike protein (specific for IgA and IgG), receptor binding domain (RBD) and nucleocapsid (N) protein for IgG in serum, and bridging ELISA with the SARS-CoV-2 RBD and N protein for specific Ig (IgG, IgM and IgA in human milk and serum). To assess the effect of pasteurization, human milk was exposed to Holder (HoP) and High Pressure Pasteurization (HPP). RESULTS: Human milk contained abundant SARS-CoV-2 antibodies in 83% of the proven cases and in 67% of the suspected cases. Unpasteurized milk with and without these antibodies was found to be capable of neutralizing a pseudovirus of SARS-CoV-2 in (97% and 85% of the samples respectively). After pasteurization, total IgA antibody levels were affected by HoP, while SARS-CoV-2 specific antibody levels were affected by HPP. Pseudovirus neutralizing capacity of the human milk samples was only retained with the HPP approach. No correlation was observed between milk antibody levels and neutralization capacity. CONCLUSIONS: Human milk from recovered COVID-19-infected mothers contains SARS-CoV-2 specific antibodies which maintained neutralization capacity after HPP. All together this may represent a safe and effective immunization strategy after HPP.

[Evaluation of the Diagnostic Performance of Different Principles of SARS-CoV-2 Commercial Antibody Tests in COVID-19 Patients]. / SARS-CoV-2 ile Ilgili Farkli Prensipli Ticari Antikor Testlerinin COVID-19 Hastalarindaki Tanisal Performanslarinin Degerlendirilmesi.

Following the emergence of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) and using only PCR for diagnosis, antibody tests have been rapidly developed by various commercial companies. There are differences between the sensitivity and specificity of these tests due to the usage of different viral target proteins and antibody subclasses. In order to evaluate the diagnostic use of these tests, we aimed to examine the diagnostic performance, especially sensitivity and specificity, of SARS-CoV-2 IgM, IgA and IgG tests of various companies (Abbott, Roche, Euroimmun, Dia.Pro, Anshlabs, Vircell, UnScience and RedCell), which have different principles (ECLIA/CLIA, EIA, LFA). Current (n= 180) and past (n= 180) COVID-19 patients with clinical and molecular diagnosis of COVID-19 admitted to Istanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine Hospital, Pandemic Polyclinic with suspected COVID-19 infection, were included in our study. The patients admitted within the first 3 weeks after the onset of symptoms were included in the current patient group, and those admitted at the third and after the third week were included in the past patient group. Serum samples (n= 180) obtained from Istanbul Sisli Hamidiye Etfal Training and Research Hospital, Blood Center between April and June 2018 before the COVID-19 pandemic were included in the study as a control group. All the tests included in our study were studied with the recommendations of the manufacturer companies. Between the IgG detection tests with different principles in patients with past COVID-19, the sensitivity and specificity values of the most effective tests were; 86.7%/99.4% (Abbott), 86.1%/98.9% (Dia.Pro), 91.3%/95% (RedCell). Between the IgM detection tests with different principles in current COVID-19 patients, the sensitivity and specificity values were; 67.8%/99.4% (Abbott), 68.9%/98.6% (Vircell), 50%/97.5% (RedCell). Abbott IgM with a kappa coefficient of 0.67 and Vircell IgM + IgA test with a kappa coefficient of 0.65 showed the best fit in patients with current COVID-19 infection. In patients with past COVID-19, Abbott IgG with 0.86 kappa coefficient and Dia.Pro IgG test with 0.85 kappa coefficient showed the best match. Due to the low sensitivity of IgM detection antibody tests, they should not be preferred instead of real-time reverse transcriptase polymerase chain reaction in routine diagnosis. IgG detection tests may be preferred to detect the antibody response and the titers in people who have had COVID-19 for population seroprevalence and especially therapeutic immune plasma production. However, it is thought that the combined use of both ECLIA/CLIA-based and EIA/ELISA-based tests together may be more effective in routine use for SARS-CoV-2 IgG tests.

SARS-CoV-2 Antigens Expressed in Plants Detect Antibody Responses in COVID-19 Patients.

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has swept the world and poses a significant global threat to lives and livelihoods, with 115 million confirmed cases and at least 2.5 million deaths from Coronavirus disease 2019 (COVID-19) in the first year of the pandemic. Developing tools to measure seroprevalence and understand protective immunity to SARS-CoV-2 is a priority. We aimed to develop a serological assay using plant-derived recombinant viral proteins, which represent important tools in less-resourced settings. Methods: We established an indirect ELISA using the S1 and receptor-binding domain (RBD) portions of the spike protein from SARS-CoV-2, expressed in Nicotiana benthamiana. We measured antibody responses in sera from South African patients (n = 77) who had tested positive by PCR for SARS-CoV-2. Samples were taken a median of 6 weeks after the diagnosis, and the majority of participants had mild and moderate COVID-19 disease. In addition, we tested the reactivity of pre-pandemic plasma (n = 58) and compared the performance of our in-house ELISA with a commercial assay. We also determined whether our assay could detect SARS-CoV-2-specific IgG and IgA in saliva. Results: We demonstrate that SARS-CoV-2-specific immunoglobulins are readily detectable using recombinant plant-derived viral proteins, in patients who tested positive for SARS-CoV-2 by PCR. Reactivity to S1 and RBD was detected in 51 (66%) and 48 (62%) of participants, respectively. Notably, we detected 100% of samples identified as having S1-specific antibodies by a validated, high sensitivity commercial ELISA, and optical density (OD) values were strongly and significantly correlated between the two assays. For the pre-pandemic plasma, 1/58 (1.7%) of samples were positive, indicating a high specificity for SARS-CoV-2 in our ELISA. SARS-CoV-2-specific IgG correlated significantly with IgA and IgM responses. Endpoint titers of S1- and RBD-specific immunoglobulins ranged from 1:50 to 1:3,200. S1-specific IgG and IgA were found in saliva samples from convalescent volunteers. Conclusion: We demonstrate that recombinant SARS-CoV-2 proteins produced in plants enable robust detection of SARS-CoV-2 humoral responses. This assay can be used for seroepidemiological studies and to measure the strength and durability of antibody responses to SARS-CoV-2 in infected patients in our setting.

Chlorogenic acid is a positive regulator of MDA5, TLR7 and NF-κB signaling pathways mediated antiviral responses against Gammacoronavirus infection.

Chlorogenic acid (CGA) is a phenolic compound that has been well studied for its antiviral, anti-inflammatory and immune stimulating properties. This research was aimed to focus on the antiviral properties of CGA on infectious bronchitis virus (IBV) in vivo and in vitro for the very first time. The outcome of in vitro experiments validated that, out of five previously reported antiviral components, CGA significantly reduced the relative mRNA expression of IBV-N in CEK cells. At high concentration (400 mg/kg), CGA supplementation reduced IBV-N mRNA expression levels and ameliorated the injury in trachea and lungs. The mRNA expression levels of IL-6, IL-1ß, IL-12, and NF-κB were considerably turned down, but IL-22 and IL-10 were enhanced in trachea. However, CGA-H treatment had considerably increased the expression levels of MDA5, MAVS, TLR7, MyD88, IRF7, IFN-ß and IFN-α both in trachea and lungs. Moreover, CGA-H notably induced the CD3+, CD3+ CD4+ and CD4+/CD8+ proliferation and significantly increased the IgA, IgG, and IgM levels in the serum. In conclusion, these results showed that at high concentration CGA is a strong anti-IBV compound that can effectively regulate the innate immunity through MDA5, TLR7 and NF-κB signaling pathways and have the potential to induce the cell mediated and humoral immune response in IBV infected chickens.

Role of Immunoglobulin M and A Antibodies in the Neutralization of Severe Acute Respiratory Syndrome Coronavirus 2.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions of people globally. Virus infection requires the receptor-binding domain (RBD) of the spike protein. Although studies have demonstrated anti-spike and -RBD antibodies to be protective in animal models, and convalescent plasma as a promising therapeutic option, little is known about immunoglobulin isotypes capable of blocking infection. METHODS: We studied spike- and RBD-specific immunoglobulin isotypes in convalescent and acute plasma/serum samples using a multiplex bead assay. We also determined virus neutralization activities in plasma and serum samples, and purified immunoglobulin fractions using a vesicular stomatitis pseudovirus assay. RESULTS: Spike- and RBD-specific immunoglobulin (Ig) M, IgG1, and IgA1 were produced by all or nearly all subjects at variable levels and detected early after infection. All samples displayed neutralizing activity. Regression analyses revealed that IgM and IgG1 contributed most to neutralization, consistent with IgM and IgG fractions' neutralization potency. IgA also exhibited neutralizing activity, but with lower potency. CONCLUSION: IgG, IgM, and IgA are critical components of convalescent plasma used for treatment of coronavirus disease 2019 (COVID-19).

Role of rapid antibody and ELISA tests in the evaluation of serological response in patients with SARS-CoV-2 PCR positivity.

From 7 to 8 days after the onset of symptoms in COVID-19 infection, the sensitivity of serological tests was found to be higher than that of nucleic acid tests. The aims of this study were to investigate antibody levels in patients with SARS-CoV-2 infection, to examine the relationship between antibody levels and virus load, and to evaluate the performance of 2 different commercial kits. A total of 103 patients with confirmed SARS-CoV-2 infection were included in the study. Antibodies against SARS-CoV-2 in serum samples taken from patients were investigated simultaneously with anti-SARS-CoV-2 IgG and IgA ELISAs (Euroimmun) and COVID-19 (SARS-CoV-2) IgG/IgM (Deep Blue) kits. No positivity was detected with any of the test kits in 18 (17.4%) of the 103 samples. In symptomatic patients, 100% of IgM and IgA tests were found to be positive in the group sampled after 10 days, while 100% of IgG tests were found positive after 20 days. The sensitivity of the Deep Blue COVID-19 IgG antibody kit was calculated as 81.48% and the specificity was 97.96%. While there was no statistically significant difference between the PCR CT and ELISA OD values, a positive correlation was found between the ELISA OD values and the days since the date of symptom initiation. The sensitivity and specificity of the rapid antibody test used in this study were found to be quite high. In conditions where ELISA tests cannot be applied, it is thought that it can give an idea in terms of the presence of antibodies as a simple and fast test. Although ELISA tests are valuable in the diagnosis of COVID-19 during the acute period, they are tests that can be used safely in the diagnosis of previous infections and seroepidemiological studies.

Evaluation of 18 commercial serological assays for the detection of antibodies against SARS-CoV-2 in paired serum samples.

A variety of serological tests have been developed to detect the presence of antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated the performance of 18 commercially available SARS-CoV-2 antibody assays. Early (6-8 days after the start of symptoms) and late sera (>14 days) from ICU patients (n=10 and n=16, respectively) and healthcare workers (n=5 and n=9, respectively) were included. Additionally, 22 sera were included to detect potential cross-reactivity. Test characteristics were determined for the 18 assays. In >14 days samples, the Vircell IgG and Wantai Ig ELISAs had superior sensitivity compared to the other ELISAs (96%). Furthermore, the Roche Ig, the Epitope Diagnostics IgM, Wantai IgM, Euroimmun IgG, and IgA all showed a specificity of 100%. The POCTs of Boson Biotech and ACRO Biotech showed the highest sensitivities: 100% and 96% (83.5-99.8), respectively. The POCT of Orient Gene Biotech, VOMED Diagnostics, and Coris-Bioconcept showed highest specificities (100%). For the IgM and IgA assays, the Euroimmun IgA test showed the highest sensitivity in early samples: 46.7% (23.5-70.9) to 53.3% (29.1-76.5). In general, all tests performed better in patients with severe symptoms (ICU patients). We conclude that the Wantai Ig and Vircell IgG ELISAs may be suitable for diagnostic purposes. The IgM/IgA tests performed poorer than their IgG/Ig counterparts but may have a role in diagnoses of SARS-CoV-2 in a population in which the background seroprevalence of IgG high, and IgM and/or IgA may distinguish between acute or past infection.

The real life performance of 7 automated anti-SARS-CoV-2 IgG and IgM/IgA immunoassays.

OBJECTIVES: This study was aimed at providing some insights into the real-life performance of the commercial, clinically validated anti-SARS-CoV-2 antibody assays. METHODS: The residual, anonymized samples from 97 patients referred for anti-SARS-CoV-2 antibodies testing were included in the study. The initial assessment was performed with the Euroimmun ELISAs, followed by the assays provided by: NovaTec, Snibe, Vircell, Roche, Abbott and DiaSorin. The analyses of the results were performed separately for the antibodies of the early (IgM/IgA) and late (IgG) immune response. RESULTS: We observed a high variability of the results obtained with the investigated immunoassays. The fully concordant results were reported for only 57 out of 97 samples tested for IgG antibodies and for 34 out of 97 samples for IgM/IgA. The highest percentage of positive results was noted for the Euroimmun and Vircell ELISAs and the lowest for Novatec ELISAs.We proposed to distinguish true and false positive results based on the sum of positive results obtained with different methods. We arbitrarily considered reference positive samples reactive in at least half of the assays. The assay that proved to correlate the best with those reference results was the Roche electrochemiluminescence immunoassay. CONCLUSIONS: The differences observed between immunoassays targeting the early phase antibodies were much more pronounced than between IgG assays, suggesting their lower value for clinical use. Our study also showed a high percentage of plausibly false (positive or negative) results obtained with ELISAs, which suggests their inferiority to the automated immunoassays.

Recent Advances in the Evaluation of Serological Assays for the Diagnosis of SARS-CoV-2 Infection and COVID-19.

Introduction: Few data on the diagnostic performance of serological tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are currently available. We evaluated sensitivity and specificity of five different widely used commercial serological assays for the detection of SARS-CoV-2-specific IgG, IgM, and IgA antibodies using reverse transcriptase-PCR assay in nasopharyngeal swab as reference standard test. Methods: A total of 337 plasma samples collected in the period April-June 2020 from SARS-CoV-2 RT-PCR positive (n = 207) and negative (n = 130) subjects were investigated by one point-of-care lateral flow immunochromatographic assay (LFIA IgG and IgM, Technogenetics) and four fully automated assays: two chemiluminescence immunoassays (CLIA-iFlash IgG and IgM, Shenzhen YHLO Biotech and CLIA-LIAISON® XL IgG, DiaSorin), one electrochemiluminescence immunoassay (ECLIA-Elecsys® total predominant IgG, Roche), and one enzyme-linked immunosorbent assay (ELISA IgA, Euroimmune). Results: The overall sensitivity of all IgG serological assays was >80% and the specificity was >97%. The sensitivity of IgG assays was lower within 2 weeks from the onset of symptoms ranging from 70.8 to 80%. The LFIA and CLIA-iFlash IgM showed an overall low sensitivity of 47.6 and 54.6%, while the specificity was 98.5 and 96.2%, respectively. The ELISA IgA yielded a sensitivity of 84.3% and specificity of 81.7%. However, the ELISA IgA result was indeterminate in 11.7% of cases. Conclusions: IgG serological assays seem to be a reliable tool for the retrospective diagnosis of SARS-CoV-2 infection. IgM assays seem to have a low sensitivity and IgA assay is limited by a substantial rate of indeterminate results.

RTS,S/AS01E malaria vaccine induces IgA responses against CSP and vaccine-unrelated antigens in African children in the phase 3 trial.

BACKGROUND: The evaluation of immune responses to RTS,S/AS01 has traditionally focused on immunoglobulin (Ig) G antibodies that are only moderately associated with protection. The role of other antibody isotypes that could also contribute to vaccine efficacy remains unclear. Here we investigated whether RTS,S/AS01E elicits antigen-specific serum IgA antibodies to the vaccine and other malaria antigens, and we explored their association with protection. METHODS: Ninety-five children (age 5-17 months old at first vaccination) from the RTS,S/AS01E phase 3 clinical trial who received 3 doses of RTS,S/AS01E or a comparator vaccine were selected for IgA quantification 1 month post primary immunization. Two sites with different malaria transmission intensities (MTI) and clinical malaria cases and controls, were included. Measurements of IgA against different constructs of the circumsporozoite protein (CSP) vaccine antigen and 16 vaccine-unrelated Plasmodium falciparum antigens were performed using a quantitative suspension array assay. RESULTS: RTS,S vaccination induced a 1.2 to 2-fold increase in levels of serum/plasma IgA antibodies to all CSP constructs, which was not observed upon immunization with a comparator vaccine. The IgA response against 13 out of 16 vaccine-unrelated P. falciparum antigens also increased after vaccination, and levels were higher in recipients of RTS,S than in comparators. IgA levels to malaria antigens before vaccination were more elevated in the high MTI than the low MTI site. No statistically significant association of IgA with protection was found in exploratory analyses. CONCLUSIONS: RTS,S/AS01E induces IgA responses in peripheral blood against CSP vaccine antigens and other P. falciparum vaccine-unrelated antigens, similar to what we previously showed for IgG responses. Collectively, data warrant further investigation of the potential contribution of vaccine-induced IgA responses to efficacy and any possible interplay, either synergistic or antagonistic, with protective IgG, as identifying mediators of protection by RTS,S/AS01E immunization is necessary for the design of improved second-generation vaccines. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov: NCT008666191.

Antibody tests for identification of current and past infection with SARS-CoV-2.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and resulting COVID-19 pandemic present important diagnostic challenges. Several diagnostic strategies are available to identify current infection, rule out infection, identify people in need of care escalation, or to test for past infection and immune response. Serology tests to detect the presence of antibodies to SARS-CoV-2 aim to identify previous SARS-CoV-2 infection, and may help to confirm the presence of current infection. OBJECTIVES: To assess the diagnostic accuracy of antibody tests to determine if a person presenting in the community or in primary or secondary care has SARS-CoV-2 infection, or has previously had SARS-CoV-2 infection, and the accuracy of antibody tests for use in seroprevalence surveys. SEARCH METHODS: We undertook electronic searches in the Cochrane COVID-19 Study Register and the COVID-19 Living Evidence Database from the University of Bern, which is updated daily with published articles from PubMed and Embase and with preprints from medRxiv and bioRxiv. In addition, we checked repositories of COVID-19 publications. We did not apply any language restrictions. We conducted searches for this review iteration up to 27 April 2020. SELECTION CRITERIA: We included test accuracy studies of any design that evaluated antibody tests (including enzyme-linked immunosorbent assays, chemiluminescence immunoassays, and lateral flow assays) in people suspected of current or previous SARS-CoV-2 infection, or where tests were used to screen for infection. We also included studies of people either known to have, or not to have SARS-CoV-2 infection. We included all reference standards to define the presence or absence of SARS-CoV-2 (including reverse transcription polymerase chain reaction tests (RT-PCR) and clinical diagnostic criteria). DATA COLLECTION AND ANALYSIS: We assessed possible bias and applicability of the studies using the QUADAS-2 tool. We extracted 2x2 contingency table data and present sensitivity and specificity for each antibody (or combination of antibodies) using paired forest plots. We pooled data using random-effects logistic regression where appropriate, stratifying by time since post-symptom onset. We tabulated available data by test manufacturer. We have presented uncertainty in estimates of sensitivity and specificity using 95% confidence intervals (CIs). MAIN RESULTS: We included 57 publications reporting on a total of 54 study cohorts with 15,976 samples, of which 8526 were from cases of SARS-CoV-2 infection. Studies were conducted in Asia (n = 38), Europe (n = 15), and the USA and China (n = 1). We identified data from 25 commercial tests and numerous in-house assays, a small fraction of the 279 antibody assays listed by the Foundation for Innovative Diagnostics. More than half (n = 28) of the studies included were only available as preprints. We had concerns about risk of bias and applicability. Common issues were use of multi-group designs (n = 29), inclusion of only COVID-19 cases (n = 19), lack of blinding of the index test (n = 49) and reference standard (n = 29), differential verification (n = 22), and the lack of clarity about participant numbers, characteristics and study exclusions (n = 47). Most studies (n = 44) only included people hospitalised due to suspected or confirmed COVID-19 infection. There were no studies exclusively in asymptomatic participants. Two-thirds of the studies (n = 33) defined COVID-19 cases based on RT-PCR results alone, ignoring the potential for false-negative RT-PCR results. We observed evidence of selective publication of study findings through omission of the identity of tests (n = 5). We observed substantial heterogeneity in sensitivities of IgA, IgM and IgG antibodies, or combinations thereof, for results aggregated across different time periods post-symptom onset (range 0% to 100% for all target antibodies). We thus based the main results of the review on the 38 studies that stratified results by time since symptom onset. The numbers of individuals contributing data within each study each week are small and are usually not based on tracking the same groups of patients over time. Pooled results for IgG, IgM, IgA, total antibodies and IgG/IgM all showed low sensitivity during the first week since onset of symptoms (all less than 30.1%), rising in the second week and reaching their highest values in the third week. The combination of IgG/IgM had a sensitivity of 30.1% (95% CI 21.4 to 40.7) for 1 to 7 days, 72.2% (95% CI 63.5 to 79.5) for 8 to 14 days, 91.4% (95% CI 87.0 to 94.4) for 15 to 21 days. Estimates of accuracy beyond three weeks are based on smaller sample sizes and fewer studies. For 21 to 35 days, pooled sensitivities for IgG/IgM were 96.0% (95% CI 90.6 to 98.3). There are insufficient studies to estimate sensitivity of tests beyond 35 days post-symptom onset. Summary specificities (provided in 35 studies) exceeded 98% for all target antibodies with confidence intervals no more than 2 percentage points wide. False-positive results were more common where COVID-19 had been suspected and ruled out, but numbers were small and the difference was within the range expected by chance. Assuming a prevalence of 50%, a value considered possible in healthcare workers who have suffered respiratory symptoms, we would anticipate that 43 (28 to 65) would be missed and 7 (3 to 14) would be falsely positive in 1000 people undergoing IgG/IgM testing at days 15 to 21 post-symptom onset. At a prevalence of 20%, a likely value in surveys in high-risk settings, 17 (11 to 26) would be missed per 1000 people tested and 10 (5 to 22) would be falsely positive. At a lower prevalence of 5%, a likely value in national surveys, 4 (3 to 7) would be missed per 1000 tested, and 12 (6 to 27) would be falsely positive. Analyses showed small differences in sensitivity between assay type, but methodological concerns and sparse data prevent comparisons between test brands. AUTHORS' CONCLUSIONS: The sensitivity of antibody tests is too low in the first week since symptom onset to have a primary role for the diagnosis of COVID-19, but they may still have a role complementing other testing in individuals presenting later, when RT-PCR tests are negative, or are not done. Antibody tests are likely to have a useful role for detecting previous SARS-CoV-2 infection if used 15 or more days after the onset of symptoms. However, the duration of antibody rises is currently unknown, and we found very little data beyond 35 days post-symptom onset. We are therefore uncertain about the utility of these tests for seroprevalence surveys for public health management purposes. Concerns about high risk of bias and applicability make it likely that the accuracy of tests when used in clinical care will be lower than reported in the included studies. Sensitivity has mainly been evaluated in hospitalised patients, so it is unclear whether the tests are able to detect lower antibody levels likely seen with milder and asymptomatic COVID-19 disease. The design, execution and reporting of studies of the accuracy of COVID-19 tests requires considerable improvement. Studies must report data on sensitivity disaggregated by time since onset of symptoms. COVID-19-positive cases who are RT-PCR-negative should be included as well as those confirmed RT-PCR, in accordance with the World Health Organization (WHO) and China National Health Commission of the People's Republic of China (CDC) case definitions. We were only able to obtain data from a small proportion of available tests, and action is needed to ensure that all results of test evaluations are available in the public domain to prevent selective reporting. This is a fast-moving field and we plan ongoing updates of this living systematic review.

Comparison of six commercially available SARS-CoV-2 antibody assays-Choice of assay depends on intended use.

OBJECTIVES: Evaluate six commercial serological assays for detection of IgA, IgM or IgG SARS-CoV-2 antibodies in different disease severities. METHODS: Three lateral flow tests (LFTs) (Acro IgM/IgG, CTK IgM/IgG, Livzon IgM/IgG) and three ELISA assays (Euroimmun IgA and IgG, Wantai IgM) were included. Application was evaluated using samples from 57 patients with a positive SARS-CoV-2 reverse transcription polymerase chain reaction, stratified according to disease severity. Specificity was assessed using historical samples from 200 blood donors. RESULTS: While IgM LFTs failed to detect SARS-CoV-2 antibodies in 37-84% of non-hospitalised patients, the Wantai IgM ELISA detected antibodies in 79%. The Euroimmun IgG ELISA detected antibodies in 95% of non-hospitalised patients. IgA, IgM and IgG ELISA levels were initially low, increased over time, and correlated with disease severity. LFT sensitivity declined in samples taken >28 days after symptom onset/resolution. The Livzon IgG LFT had the highest specificity (98.5%), followed by the Euroimmun IgG ELISA (96.2%). The specificity for Euroimmun IgA ELISA improved (≥97.5%) using a custom cut-off value (4.0). CONCLUSIONS: The sensitive and semi-quantitative ELISA assays are most appropriate for serologic detection of SARS-CoV-2 infection in mild cases. Livzon LFT and Euroimmun ELISA had the highest specificity among the IgG assays, making them most suitable for seroprevalence studies.

Anti-SARS-Cov-2 IgA Response in Tears of COVID-19 Patients.

The pandemic virus SARS-CoV-2 has been reported to be able to enter the body via the eye conjunctiva, but the presence of antiviral response in the eye remains poorly known. Our study was thus aimed to analyze the presence of secretory mucosal anti-SARS-CoV-2 type A immunoglobulins (IgA) in the conjunctival fluid of COVID-19 patients. The tears of 28 COVID-19 patients and 20 uninfected controls were collected by the Schirmer test and analyzed by a specific ELISA assay detecting anti-spike (S1) virus protein IgA. The results showed that 35.7% of COVID-19 subjects have specific antiviral IgA at the ocular level, persisting till 48 days post disease onset. Most of the IgA positive subjects presented mild symptoms. The collected data indicate a prolonged persistence of anti-SARS-CoV-2 IgA at the eye level and suggest that IgA detection may be extremely helpful in clarifying virus pathology and epidemiology.

IgA dominates the early neutralizing antibody response to SARS-CoV-2.

Humoral immune responses are typically characterized by primary IgM antibody responses followed by secondary antibody responses associated with immune memory and composed of IgG, IgA, and IgE. Here, we measured acute humoral responses to SARS-CoV-2, including the frequency of antibody-secreting cells and the presence of SARS-CoV-2-specific neutralizing antibodies in the serum, saliva, and bronchoalveolar fluid of 159 patients with COVID-19. Early SARS-CoV-2-specific humoral responses were dominated by IgA antibodies. Peripheral expansion of IgA plasmablasts with mucosal homing potential was detected shortly after the onset of symptoms and peaked during the third week of the disease. The virus-specific antibody responses included IgG, IgM, and IgA, but IgA contributed to virus neutralization to a greater extent compared with IgG. Specific IgA serum concentrations decreased notably 1 month after the onset of symptoms, but neutralizing IgA remained detectable in saliva for a longer time (days 49 to 73 post-symptoms). These results represent a critical observation given the emerging information as to the types of antibodies associated with optimal protection against reinfection and whether vaccine regimens should consider targeting a potent but potentially short-lived IgA response.