Question: are there serological tests that detect antibodies to coronavirus?

Title: Seroepidemiologic Study Designs for Determining SARS-COV-2 Transmission and Immunity Content: Serologic studies are crucial for clarifying dynamics of the coronavirus disease pandemic. Past work on serologic studies (e.g., during influenza pandemics) has made relevant contributions, but specific conditions of the current situation require adaptation. Although detection of antibodies to measure exposure, immunity, or both seems straightforward conceptually, numerous challenges exist in terms of sample collection, what the presence of antibodies actually means, and appropriate analysis and interpretation to account for test accuracy and sampling biases. Successful deployment of serologic studies depends on type and performance of serologic tests, population studied, use of adequate study designs, and appropriate analysis and interpretation of data. We highlight key questions that serologic studies can help answer at different times, review strengths and limitations of different assay types and study designs, and discuss methods for rapid sharing and analysis of serologic data to determine global transmission of severe acute respiratory syndrome coronavirus 2.

Title: Seroepidemiologic Study Designs for Determining SARS-COV-2 Transmission and Immunity. Content: Serologic studies are crucial for clarifying dynamics of the coronavirus disease pandemic. Past work on serologic studies (e.g., during influenza pandemics) has made relevant contributions, but specific conditions of the current situation require adaptation. Although detection of antibodies to measure exposure, immunity, or both seems straightforward conceptually, numerous challenges exist in terms of sample collection, what the presence of antibodies actually means, and appropriate analysis and interpretation to account for test accuracy and sampling biases. Successful deployment of serologic studies depends on type and performance of serologic tests, population studied, use of adequate study designs, and appropriate analysis and interpretation of data. We highlight key questions that serologic studies can help answer at different times, review strengths and limitations of different assay types and study designs, and discuss methods for rapid sharing and analysis of serologic data to determine global transmission of severe acute respiratory syndrome coronavirus 2.

Title: Prevalence of antibody to human coronaviruses 229E, OC43 and neonatal calf diarrhea coronavirus (NCDCV) in patients of Northern Italy Content: A seroepidemiological study for detection of antibody to human coronaviruses OC43, 229E, and neonatal calf diarrhea coronavirus (NCDCV), has been carried out using sera collected from hospitalized patients or healthy persons through routine laboratory tests in Northern Italy. Patients tested were children and adults with different pathological diseases. Antibody detection was performed by using an indirect immunoperoxidase staining technique (for all viruses) and, in the case of OC43 and NCDCV, antibody detection was obtained even with a hemagglutination inhibition test and a plaque reduction neutralization assay. Results obtained show a significant difference in the prevalence of antibody to 229E between children and adult group. Furthermore, a different titer was observed, within the two groups, between patients affected by hematological diseases (leukemia) and patients with other diseases. Finally, our data seem to confirm previous studies reporting a very high prevalence of antibody to coronavirus OC43 but a less detectable seropositivity to coronavirus 229E.

Title: Coronavirus antibody detection in cats by computer-assisted kinetics-based enzyme-linked immunosorbent assay (KELA): field studies. Content: A total of 2238 feline serum samples submitted to the New York State Diagnostic Laboratory over a 1-year period were tested for the presence of coronavirus antibodies, using the computer-assisted, kinetics-based enzyme-linked immunosorbent assay (KELA). Cats from which sera were obtained were categorized by sex, age, breed, and disease status, and variations in mean antibody titers for different sub-classifications within each category were analyzed by computerized statistical analysis. As expected, higher mean antibody titers were recorded for cats with feline infectious peritonitis, and for cats with a recent history of possible coronavirus exposure. However, an unexpected inverse relationship between coronavirus antibody titer and age was also found. Certain cattery-oriented pure breeds appeared to have higher mean antibody titers, because their sample populations contained a higher percentage of younger cats and cats of unknown age-groups which, over-all, had higher mean titers. Taken together, the data substantiated the efficacy of the computer-assisted KELA for routine detection of serum coronavirus antibodies in cats.

Title: Reliability of serological tests for COVID-19: Comparison of three immunochromatography test kits for SARS-CoV-2 antibodies. Content: Background: Several immunochromatographic serological test kits have been developed to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific antibodies, but their relative performance and potential clinical utility is unclear. Methods: Three commercially available serological test kits were evaluated using 99 serum samples collected from 29 patients diagnosed with coronavirus disease 2019 (COVID-19). Results: The IgM antibody-positive rates of the three serological test kits for samples taken at the early stage of the disease (0-6 days after onset) were 19.0%, 23.8%, and 19.0%, respectively. The IgM antibody-positive rates over the entire period were 21.2%, 60.6%, and 15.2%, respectively. The IgG antibody-positive rates for samples taken after 13 days of onset were 100.0%, 97.6%, and 97.6%, respectively. Conclusion: There were large differences among the results of the three test kits. Only few cases showed positive results for IgM in the early stage of disease and the IgM antibody-positive rates over the entire period were low, suggesting that the kits used in this study were unsuitable for diagnosis of COVID-19. The IgG antibody was positive in almost all samples after 13 days of onset, suggesting that it may be useful for determining infections in the recent past.

Title: Prevalence of coronavirus antibodies in rabbits. Content: Antibodies to coronavirus were detected by an indirect fluorescent antibody test in rabbit sera from six rabbitries. The prevalence ranged from 3 to 40% in different rabbitries and most seropositive rabbits were more than 4 months old. A rabbitry with high prevalence of antibodies and high incidence of diarrhea could serve as a source of virus and aid in studying the natural history of coronavirus infection in rabbits.

Title: SEROEPIDEMIOLOGIC STUDIES OF CORONAVIRUS INFECTION IN ADULTS AND CHILDREN(1) Content: McIntosh, K. A. Z. Kapikian, H. C Turner, J. W. Hartley, R. H. Parrott and R. M. Chanock. (Lab. of Infectious Diseases, NIAID, NIH, Bethesda, Md. 20014) Sero-epidemiologic studies of coronavirus infection in adults and children. Amer. J. Epid., 1970, 97: 585–592-A seroepidemiologic study of infection by coronavirus strains 229E, OC38, OC43, and mouse hepatitis virus (MHV) strain A-59, is described. In adults with upper respiratory disease, two “outbreaks” of coronavirus infection occurred, one during the winter of 1965–1966 associated with complement fixing (CF) antibody responses to OC38, OC43 and MHV, and the other during the following winter associated with CF antibody responses to 229E. In hospitalized children, infection with 229E was rare; infection with OC38, OC43, and MHV occurred less often in hospitalized children with lower respiratory tract disease (3.5%) than in a control group with non-respiratory tract disease (8.2%). The limitations of the CF test using available coronavirus antigens are discussed.

Title: SEROEPIDEMIOLOGIC SURVEY OF CORONAVIRUS (STRAIN 229E) INFECTIONS IN A POPULATION OF CHILDREN Content: The indirect hemagglutination (IHA) test for coronavirus 229E antibodies was used for serodiagnostic and seroepidemiologic studies in a population of children. Subjects ranged in age from 5 to 19 years and lived in a home which participated in a longitudinal surveillance of respiratory illness (1960–1968). During this period 1477 respiratory illnesses were observed; 63 (4%) were associated with sero-response (fourfold or greater antibody rises) to 229E. An additional 105 sero-responses were associated with unreported or subclinical illness. The frequency of these infections was cyclical, and 229E and coronavirus OC 43 infections peaked in different years among the same population. Sero-responses occurred mainly in the fall, winter and spring quarters. Preexisting antibody was demonstrated in one-third of the children with 229E seroresponses. Clinical studies indicated that the most frequent complaints with 229E infections were sore throat, coryza and cough, and the most frequent findings were pharyngeal injection, coryza and fever.

Title: Review of Current Advances in Serologic Testing for COVID-19 Content: OBJECTIVES: To examine and summarize the current literature on serologic methods for the detection of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: A literature review was performed using searches in databases including PubMed, medRxiv, and bioRxiv. Thirty-two peer-reviewed papers and 23 preprints were examined. RESULTS: The studies included lateral flow immunoassay, enzyme-linked immunosorbent assay, chemiluminescence immunoassay, and neutralizing antibody assays. The use of all major SARS-CoV-2 antigens was demonstrated to have diagnostic value. Assays measuring total antibody reactivity had the highest sensitivity. In addition, all the methods provided opportunities to characterize the humoral immune response by isotype. The combined use of IgM and IgG detection resulted in a higher sensitivity than that observed when detecting either isotype alone. Although IgA was rarely studied, it was also demonstrated to be a sensitive marker of infection, and levels correlated with disease severity and neutralizing activity. CONCLUSIONS: The use of serologic testing, in conjunction with reverse transcription polymerase chain reaction testing, was demonstrated to significantly increase the sensitivity of detection of patients infected with SARS-CoV-2. There was conflicting evidence regarding whether antibody titers correlated with clinical severity. However, preliminary investigations indicated some immunoassays may be a surrogate for the prediction of neutralizing antibody titers and the selection of recovered patients for convalescent serum donation.

Title: Serodiagnostics for Severe Acute Respiratory Syndrome-Related Coronavirus-2: A Narrative Review Content: Accurate serologic tests to detect host antibodies to severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) will be critical for the public health response to the coronavirus disease 2019 pandemic. Many use cases are envisaged, including complementing molecular methods for diagnosis of active disease and estimating immunity for individuals. At the population level, carefully designed seroepidemiologic studies will aid in the characterization of transmission dynamics and refinement of disease burden estimates and will provide insight into the kinetics of humoral immunity. Yet, despite an explosion in the number and availability of serologic assays to test for antibodies against SARS-CoV-2, most have undergone minimal external validation to date. This hinders assay selection and implementation, as well as interpretation of study results. In addition, critical knowledge gaps remain regarding serologic correlates of protection from infection or disease, and the degree to which these assays cross-react with antibodies against related coronaviruses. This article discusses key use cases for SARS-CoV-2 antibody detection tests and their application to serologic studies, reviews currently available assays, highlights key areas of ongoing research, and proposes potential strategies for test implementation.

Title: Serodiagnostics for Severe Acute Respiratory Syndrome–Related Coronavirus-2: A Narrative Review Content: Accurate serologic tests to detect host antibodies to severe acute respiratory syndrome–related coronavirus-2 (SARS-CoV-2) will be critical for the public health response to the coronavirus disease 2019 pandemic. Many use cases are envisaged, including complementing molecular methods for diagnosis of active disease and estimating immunity for individuals. At the population level, carefully designed seroepidemiologic studies will aid in the characterization of transmission dynamics and refinement of disease burden estimates and will provide insight into the kinetics of humoral immunity. Yet, despite an explosion in the number and availability of serologic assays to test for antibodies against SARS-CoV-2, most have undergone minimal external validation to date. This hinders assay selection and implementation, as well as interpretation of study results. In addition, critical knowledge gaps remain regarding serologic correlates of protection from infection or disease, and the degree to which these assays cross-react with antibodies against related coronaviruses. This article discusses key use cases for SARS-CoV-2 antibody detection tests and their application to serologic studies, reviews currently available assays, highlights key areas of ongoing research, and proposes potential strategies for test implementation.

Title: [Acute respiratory disease caused by coronaviruses]. Content: Serological evidence of coronavirus aetiology was found in 11 patients out of 218 persons examined (5%) in the period of 1986-1987, and in 23 cases out of 311 patients (7.4%) in the period of 1987-1988. Antibody titres with a minimum of four-time elevated values in paired sera using the ELISA method were considered conclusive. Coronaviruses 229E and OC43 were employed. During the first period, more persons were infected by coronavirus 229E, while in the second period, by coronavirus OC43. The disease was equally spread over all the months of the years. For example, in the 1987-1988 period, most infections occurred in February, when out of 60 examined patients, 9 were coronavirus positive (15%).

Title: Prevalence of canine coronavirus antibodies by an enzyme-linked immunosorbent assay in dogs in the south of Italy Content: Abstract An enzyme-linked immunosorbent assay (Elisa), using as antigen canine coronavirus-infected CrFK cell supernatant, was developed to detect antibodies against canine coronavirus (CCoV). Out of a total of 109 dog serum samples, 80 which were positive by routine virus neutralisation test were also Elisa positive. Seventeen samples which were negative by the virus neutralisation test, were positive by Elisa and by the confirmatory Western blotting test. The Elisa was substantially more sensitive than the virus neutralisation test in detecting antibodies to CCoV and may be used as an alternative technique to virus neutralisation.

Title: Are antibodies tests accurate? Understanding predictive values and uncertainty of serology tests for the novel coronavirus. Content: Antibodies testing in the coronavirus era is frequently promoted, but the underlying statistics behind their validation has come under more scrutiny in recent weeks. We provide calculations, interpretations, and plots of positive and negative predictive values under a variety of scenarios. Prevalence, sensitivity, and specificity are estimated within ranges of values from researchers and antibodies manufacturers. Illustrative examples are highlighted, and interactive plots are provided in the Supplementary Material. Implications are discussed for society overall and across diverse locations with different levels of disease burden. Specifically, the proportion of positive serology tests that are false can differ drastically from up to 3% to 88% for people from different places with different proportions of infected people in the populations while the false negative rate is typically under 10%.

Title: Severe Acute Respiratory Syndrome Coronavirus 2-Specific Antibody Responses in Coronavirus Disease Patients Content: A new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has recently emerged to cause a human pandemic. Although molecular diagnostic tests were rapidly developed, serologic assays are still lacking, yet urgently needed. Validated serologic assays are needed for contact tracing, identifying the viral reservoir, and epidemiologic studies. We developed serologic assays for detection of SARS-CoV-2 neutralizing, spike protein-specific, and nucleocapsid-specific antibodies. Using serum samples from patients with PCR-confirmed SARS-CoV-2 infections, other coronaviruses, or other respiratory pathogenic infections, we validated and tested various antigens in different in-house and commercial ELISAs. We demonstrated that most PCR-confirmed SARS-CoV-2-infected persons seroconverted by 2 weeks after disease onset. We found that commercial S1 IgG or IgA ELISAs were of lower specificity, and sensitivity varied between the 2 assays; the IgA ELISA showed higher sensitivity. Overall, the validated assays described can be instrumental for detection of SARS-CoV-2-specific antibodies for diagnostic, seroepidemiologic, and vaccine evaluation studies.

Title: Comparison of serologic techniques for the detection of antibodies against feline coronaviruses. Content: The seroprevalence of feline coronavirus (FCoV) antibodies was studied in cats in southern Italy. One hundred twenty sera collected from cats belonging to catteries or community shelters and to households were tested for FCoV type I and II antibodies. The virus neutralization (VN) was performed and compared with indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). Ninety-six sera tested positive for FCoV antibodies by VN and ELISA. Interestingly, ELISA revealed 2 more positive sera than did the VN test and 3 more positive sera than did the IFAT. All results were confirmed by Western blotting. ELISA proved to be more sensitive and detected a seroprevalence of about 82%. Considering the cross-reactivity of FCoV type I and type II, ELISA was able to detect antibodies against both serotypes, allowing the use of the assay as a reference test for sera screening. The high prevalence of antibodies observed indicates that FCoVs are common in southern Italian cat populations.

Title: Serological Tests for SARS-CoV-2 Coronavirus by Commercially Available Point-of-Care and Laboratory Diagnostics in Pre-COVID-19 Samples in Japan Content: Serological tests for SARS-CoV-2 coronavirus in pre-COVID-19 samples in Japan showed 1.5%-1.75% positives, and previous surveys might overestimate COVID-19 seroprevalence in several population of Japan. These false negatives could be excluded by combination of different diagnostics to 0.25%.

Title: Severe Acute Respiratory Syndrome Coronavirus 2−Specific Antibody Responses in Coronavirus Disease Patients Content: A new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has recently emerged to cause a human pandemic. Although molecular diagnostic tests were rapidly developed, serologic assays are still lacking, yet urgently needed. Validated serologic assays are needed for contact tracing, identifying the viral reservoir, and epidemiologic studies. We developed serologic assays for detection of SARS-CoV-2 neutralizing, spike protein–specific, and nucleocapsid-specific antibodies. Using serum samples from patients with PCR-confirmed SARS-CoV-2 infections, other coronaviruses, or other respiratory pathogenic infections, we validated and tested various antigens in different in-house and commercial ELISAs. We demonstrated that most PCR-confirmed SARS-CoV-2–infected persons seroconverted by 2 weeks after disease onset. We found that commercial S1 IgG or IgA ELISAs were of lower specificity, and sensitivity varied between the 2 assays; the IgA ELISA showed higher sensitivity. Overall, the validated assays described can be instrumental for detection of SARS-CoV-2–specific antibodies for diagnostic, seroepidemiologic, and vaccine evaluation studies.

Title: Diagnostic accuracy of serological tests for covid-19: systematic review and meta-analysis Content: OBJECTIVE: To determine the diagnostic accuracy of serological tests for coronavirus disease-2019 (covid-19). DESIGN: Systematic review and meta-analysis. DATA SOURCES: Medline, bioRxiv, and medRxiv from 1 January to 30 April 2020, using subject headings or subheadings combined with text words for the concepts of covid-19 and serological tests for covid-19. ELIGIBILITY CRITERIA AND DATA ANALYSIS: Eligible studies measured sensitivity or specificity, or both of a covid-19 serological test compared with a reference standard of viral culture or reverse transcriptase polymerase chain reaction. Studies were excluded with fewer than five participants or samples. Risk of bias was assessed using quality assessment of diagnostic accuracy studies 2 (QUADAS-2). Pooled sensitivity and specificity were estimated using random effects bivariate meta-analyses. MAIN OUTCOME MEASURES: The primary outcome was overall sensitivity and specificity, stratified by method of serological testing (enzyme linked immunosorbent assays (ELISAs), lateral flow immunoassays (LFIAs), or chemiluminescent immunoassays (CLIAs)) and immunoglobulin class (IgG, IgM, or both). Secondary outcomes were stratum specific sensitivity and specificity within subgroups defined by study or participant characteristics, including time since symptom onset. RESULTS: 5016 references were identified and 40 studies included. 49 risk of bias assessments were carried out (one for each population and method evaluated). High risk of patient selection bias was found in 98% (48/49) of assessments and high or unclear risk of bias from performance or interpretation of the serological test in 73% (36/49). Only 10% (4/40) of studies included outpatients. Only two studies evaluated tests at the point of care. For each method of testing, pooled sensitivity and specificity were not associated with the immunoglobulin class measured. The pooled sensitivity of ELISAs measuring IgG or IgM was 84.3% (95% confidence interval 75.6% to 90.9%), of LFIAs was 66.0% (49.3% to 79.3%), and of CLIAs was 97.8% (46.2% to 100%). In all analyses, pooled sensitivity was lower for LFIAs, the potential point-of-care method. Pooled specificities ranged from 96.6% to 99.7%. Of the samples used for estimating specificity, 83% (10 465/12 547) were from populations tested before the epidemic or not suspected of having covid-19. Among LFIAs, pooled sensitivity of commercial kits (65.0%, 49.0% to 78.2%) was lower than that of non-commercial tests (88.2%, 83.6% to 91.3%). Heterogeneity was seen in all analyses. Sensitivity was higher at least three weeks after symptom onset (ranging from 69.9% to 98.9%) compared with within the first week (from 13.4% to 50.3%). CONCLUSION: Higher quality clinical studies assessing the diagnostic accuracy of serological tests for covid-19 are urgently needed. Currently, available evidence does not support the continued use of existing point-of-care serological tests. STUDY REGISTRATION: PROSPERO CRD42020179452.

Title: A line immunoassay utilizing recombinant nucleocapsid proteins for detection of antibodies to human coronaviruses Content: Abstract Most coronaviruses infecting humans cause mild diseases, whereas severe acute respiratory syndrome (SARS)-associated coronavirus is an extremely dangerous pathogen. Here, we report the development of a serologic assay for detection of antibodies to human coronaviruses (HCoVs) based on recombinant nucleocapsid (N) proteins of all known pathogenic strains (229E, NL63, OC43, HKU1, SARS). The novel immunoassay is highly useful for epidemiologic surveys, where use of nucleic acid diagnostics often is limited. Purified recombinant antigens were immobilized on nitrocellulose membranes and applied in a line immunoassay, which allows rapid detection of antibodies to 5 different HCoVs in a single experiment. For assay evaluation, serum samples from persons infected with 229E or OC43 (acute/convalescent), recovered SARS patients and healthy donors were analyzed. Screening for nucleocapsid (N)-specific immunoglobulin G (IgG) in convalescent sera reached 100% sensitivity. With this new technique, we found that recently identified NL63 and HKU1 contribute significantly to the overall spectrum of coronavirus infections. Possibly, cross-reactive antibody responses were observed using 229E and OC43 serum pairs. However, the potential of this assay could clearly be demonstrated employing SARS-positive serum samples, where nonspecific binding to nucleocapsids of other HCoVs was not observed. This coronavirus strain-specific line immunoassay represents a powerful tool for serologic diagnostics.

Title: Diagnosis of human coronavirus infection by immunofluorescence: Method and application to respiratory disease in hospitalized children Content: Rabbit antisera were prepared against coronavirus strains 229E and OC43 and used successfully to detect viral antigen in epithelial cells shed from the nasopharynx of symptomatic volunteers who had received coronavirus inocula three to four days before. The same serologic reagents were applied to nasopharyngeal secretion cells obtained from 106 infants and children hospitalized with respiratory tract disease and apparently not infected with conventional respiratory viruses. No coronavirus infections were detected by this method. It appears that coronavirus OC43 or 229E infections were not common in children in Tyneside hospitals during the period of study. However, fluorescence is a useful method for detection of coronavirus infections in symptomatic human subjects.

Title: Serologic and Molecular Biologic Methods for SARS-associated Coronavirus Infection, Taiwan Content: Severe acute respiratory syndrome (SARS) has raised a global alert since March 2003. After its causative agent, SARS-associated coronavirus (SARS-CoV), was confirmed, laboratory methods, including virus isolation, reverse transcriptase–polymerase chain reaction (RT-PCR), and serologic methods, have been quickly developed. In this study, we evaluated four serologic tests ( neutralization test, enzyme-linked immunosorbent assay [ELISA], immunofluorescent assay [IFA], and immunochromatographic test [ICT]) for detecting antibodies to SARS-CoV in sera of 537 probable SARS case-patients with correlation to the RT-PCR . With the neutralization test as a reference method, the sensitivity, specificity, positive predictive value, and negative predictive value were 98.2%, 98.7%, 98.7%, and 98.4% for ELISA; 99.1%, 87.8%, 88.1% and 99.1% for IFA; 33.6%, 98.2%, 95.7%, and 56.1% for ICT, respectively. We also compared the recombinant-based western blot with the whole virus–based IFA and ELISA; the data showed a high correlation between these methods, with an overall agreement of >90%. Our results provide a systematic analysis of serologic and molecular methods for evaluating SARS-CoV infection.

Title: A serological assay to detect SARS-CoV-2 seroconversion in humans Content: SARS-Cov-2 (severe acute respiratory disease coronavirus 2), which causes Coronavirus Disease 2019 (COVID19) was first detected in China in late 2019 and has since then caused a global pandemic. While molecular assays to directly detect the viral genetic material are available for the diagnosis of acute infection, we currently lack serological assays suitable to specifically detect SARS-CoV-2 antibodies. Here we describe serological enzyme-linked immunosorbent assays (ELISA) that we developed using recombinant antigens derived from the spike protein of SARS-CoV-2. Using negative control samples representing pre-COVID 19 background immunity in the general adult population as well as samples from COVID19 patients, we demonstrate that these assays are sensitive and specific, allowing for screening and identification of COVID19 seroconverters using human plasma/serum as early as two days post COVID19 symptoms onset. Importantly, these assays do not require handling of infectious virus, can be adjusted to detect different antibody types and are amendable to scaling. Such serological assays are of critical importance to determine seroprevalence in a given population, define previous exposure and identify highly reactive human donors for the generation of convalescent serum as therapeutic. Sensitive and specific identification of coronavirus SARS-Cov-2 antibody titers may, in the future, also support screening of health care workers to identify those who are already immune and can be deployed to care for infected patients minimizing the risk of viral spread to colleagues and other patients.

Title: Prevalence of feline coronavirus antibodies in cats in Bursa province, Turkey, by an enzyme-linked immunosorbent assay Content: Feline sera from Bursa province (Turkey) were assayed for coronavirus antibody using an enzyme-linked immunosorbent assay (ELISA). The study was performed on 100 sera collected from cats belonging to catteries or community shelters and to households. The serum samples were initially tested with the virus neutralisation (VN) test and the results were then compared with the ELISA. The VN yielded 79 negative and 21 positive sera but the ELISA confirmed only 74 as negative. The ELISA-negative sera were also found to be free of feline coronoviruses-specific antibodies by Western blotting. Using the VN as the gold standard test, ELISA had a sensitivity of 100% and a specificity of 93.6%, with an overall agreement of 95%. The Kappa (κ) test indicated high association between the two tests (κ =0.86, 95% confidence interval (CI) 0.743–0.980). The positive predictive value (PPV) was 0.8, and the negative predictive value (NPV) was 0.93. The prevalence of FCoV II antibodies in the sampled population based on the gold standard was 62% (95% CI 0.44–0.77) among multi-cat environments, and 4% (95% CI 0.01–0.11) among single cat households.

Title: Diagnosis of human coronavirus infections in children using enzyme‐linked immunosorbent assay Content: An enzyme‐linked immunosorbent assay (ELISA) was developed for diagnosing human coronavirus (HCV) infections in children. One hundred and seventy seven nose swabs, throat swabs, and nasopharyngeal aspirates were collected from 30 children suffering from acute respiratory infections. These samples were tested for HCV antigens by ELISA and 28.2% of the samples were shown to be HCV positive. These results indicate that our ELISA should prove useful in the diagnosis of HCV infections in children. Further studies are in progress to extend the ELISA to detect HCVs in experimentally and naturally acquired infections in adults.

Title: Characteristics and assessment of the usefulness of serological tests in the diagnostic of infections caused by coronavirus SARS-CoV-2 on the basis of available manufacturer's data and literature review. Content: Recognized in 2019 in Wuhan, China, the new SARS-CoV-2 coronavirus is responsible for the occurrence of a global pandemic disease called COVID-19. So far, confirmation of infection is based on the detection of virus RNA in a sample taken from a person meeting the suspected case definition. However, in the laboratory diagnosis of SARS-CoV-2 infections, in addition to genetic tests, serological methods can also be used to detect specific antibodies of the IgM, IgG and IgA class produced after contact with antigens or to detect viral antigen. Currently, a number of rapid immunochromatographic, chemiluminescent and ELISA immunoassay tests developed by different manufacturers for the diagnosis of COVID-19 are available on the market. Despite this fact, so far there is no WHO or ECDC recommendations or even reliable research regarding the usefulness of serological investigations in the laboratory diagnosis of infections caused by SARS-CoV-2.

Title: Characteristics and assessment of the usefulness of serological tests in the diagnostic of infections caused by coronavirus SARS-CoV-2 on the basis of available manufacturer's data and literature review Content: Recognized in 2019 in Wuhan, China, the new SARS-CoV-2 coronavirus is responsible for the occurrence of a global pandemic disease called COVID-19. So far, confirmation of infection is based on the detection of virus RNA in a sample taken from a person meeting the suspected case definition. However, in the laboratory diagnosis of SARS-CoV-2 infections, in addition to genetic tests, serological methods can also be used to detect specific antibodies of the IgM, IgG and IgA class produced after contact with antigens or to detect viral antigen. Currently, a number of rapid immunochromatographic, chemiluminescent and ELISA immunoassay tests developed by different manufacturers for the diagnosis of COVID-19 are available on the market. Despite this fact, so far there is no WHO or ECDC recommendations or even reliable research regarding the usefulness of serological investigations in the laboratory diagnosis of infections caused by SARS-CoV-2.

Title: Laboratory Tests for COVID-19: A Review of Peer-Reviewed Publications and Implications for Clinical UIse. Content: Diagnostic tests for the coronavirus infection 2019 (COVID-19) are critical for prompt diagnosis, treatment and isolation to break the cycle of transmission. A positive real-time reverse-transcriptase polymerase chain reaction (RT-PCR), in conjunction with clinical and epidemiologic data, is the current standard for diagnosis, but several challenges still exist. Serological assays help to understand epidemiology better and to evaluate vaccine responses but they are unreliable for diagnosis in the acute phase of illness or assuming protective immunity. Serology is gaining attention, mainly because of convalescent plasma gaining importance as treatment for clinically worsening COVID-19 patients. We provide a narrative review of peer-reviewed research studies on RT-PCR, serology and antigen immune-assays for COVID-19, briefly describe their lab methods and discuss their limitations for clinical practice.

Title: Enzyme-linked immunosorbent assay for the detection of the coronavirus-like agent and its antibodies in pigs with porcine epidemic diarrhea Content: Abstract An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of the coronavirus-like agent in feces of pigs naturally affected with porcine epidemic diarrhea (PED) or experimentally infected with the CV777 isolate. The assay was specific and more sensitive than electron microscopy. An ELISA blocking assay is described for the detection and titration of antibodies. Specific antibody formation was demonstrated in pigs experimentally infected with CV777 and in swine naturally affected with PED.

Title: Seroprevalence of Antibodies to SARS-CoV-2 in Healthcare Workers in Non-epidemic Region: A Report from Iwate Prefecture in Japan Content: Background: As of June 8, 2020, Iwate is the only one of 47 prefectures in Japan with no confirmed coronavirus disease 2019 (COVID-19) cases. Serological survey for COVID-19 antibodies is crucial in area with low prevalence as well as epidemic area when addressing health and social issues caused by COVID-19. Rapid, accurate and easy-to-use antibody tests as well laboratory-based antibody tests are necessary for confirming immunity in a given community. Methods:Serum samples from healthcare workers (n = 1,000, mean 40 {+/-} 11 years) of Iwate Prefectural Central Hospital, Iwate, Japan were tested for the prevalence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies. Two laboratory-based quantitative tests (Abbott Architect SARS-CoV-2 IgG and Roche Elecsys Anti-SARS-CoV-2 assays) and one point-of-care (POC) qualitative test (Alfa Instant-view plus COVID-19 Test) were performed simultaneously. Sensitivity and specificity were 100%, 99.6% in Abbott assay; 100%, 99.8% in Roche assay; 97.8%, 94.6% in Alfa POC test, respectively. Results:The laboratory-based quantitative tests showed positive in 4 of 1,000 samples (0.4%) (95% CI: 0.01 to 0.79): 4/1,000 (0.4%) (95% CI: 0.01 to 0.79) in Abbott; 0/1,000 (0%) in Roche. Positive samples were not detected for both Abbott and Roche assays. The POC qualitative test showed positive in 33 of 1,000 samples (3.3%) (95% CI: 2.19 to 4.41), showing higher rates than those of the laboratory-based quantitative tests. There were no samples with simultaneous positive reaction for two quantitative tests and a POC test. Conclusions: Infected COVID-2 cases were not confirmed by a retrospective serological study in healthcare workers of our hospital. The POC qualitative tests with lower specificity have the potential for higher false positive reactions than the laboratory-based quantitative tests in areas with very low prevalence of COVID-19.

Title: Antigenic relationships amongst coronaviruses Content: Several serological interrelationships between various members of the corona-virus group have been revealed in neutralization, complement fixation, and gel-diffusion tests, using human and hyperimmune animal sera. Several members of this group of human and animal pathogens are shown to cross-react in one or more type of test, but one member, avian infectious bronchitis virus, was shown to be unrelated. Mouse hepatitis virus (MHV(3)) was found to be antigenically related to a number of human types of coronavirus. Difficulties were encountered in the investigation of paired human sera in demonstrating the specificity of antibody rises, placing doubt on the values of some serological studies. The significance of these interrelationships is discussed in the light of other investigations.

Title: Detection of human coronavirus 229E-specific antibodies using recombinant fusion proteins Content: Abstract Human coronaviruses are known to be a common cause of respiratory infections in man. However, the diagnosis of human coronavirus infections is not carried out routinely, primarily because the isolation and propagation of these viruses in tissue culture is difficult and time consuming. The aim of this study was to evaluate the use of recombinant, bacterial expressed proteins in the serodiagnosis of coronavirus infections. Two proteins were examined: the human coronavirus 229E nucleocapsid protein (N), expressed as a fusion protein in the vector pUR and the coronavirus 229E surface glycoprotein (S), expressed as a fusion protein in the vector pROS. The recombinant proteins were used as antigens in Western blot (WB) assays to detect the 229E-specific IgG antibodies and the results were compared with a standard serological method, indirect immunofluorescence. Serum samples of 51 paediatric patients, suffering from acute respiratory illness, and 10 adults, voluntarily infected with human coronavirus, were tested. The serum samples of the adult group had coronavirus-specific IgG antibodies in both test systems. In contrast, only 8 51 sera of the paediatric group were positive for coronavirus-specific IgG by both WB and IF and 20 51 sera were positive by WB, but not by IF. The overall incidence of human coronavirus infections in the paediatric age group was 55% evaluated by WB analysis and 16% evaluated by IF. This study shows that recombinant human coronavirus 229E proteins are suitable reagents for the epidemiological screening of coronavirus 229E infections.

Title: Coronative antibody tires in sera of healthy adults and experimentally infected volunteers. Content: Six coronaviruses isolated in the U.S.A. have been inoculated into volunteers and all produced colds. Between 10 and 20% of infected volunteers developed heterologous antibody responses after these and other experimental infections with coronaviruses. The haemagglutination-inhibition test with the OC43 virus strain was found to detect antibody rises after infection with a variety of strains.Studies on normal adult sera taken between 1965 and 1970 revealed a high frequency of neutralizing antibody to one strain (229 E) and a frequency of HI antibody to strain OC43 which fluctuated from year to year. Complement-fixing antibodies to these two viruses were also found, revealing an apparent increase in the activity of coronaviruses in the general population of the U.K., during the winter of 1968-9.

Title: Development of a nucleocapsid-based human coronavirus immunoassay and estimates of individuals exposed to coronavirus in a U.S. metropolitan population. Content: Coronaviruses cause respiratory infections ranging from common colds to severe acute respiratory syndrome (SARS) in humans. Estimates for exposure to non-SARS coronaviruses are high, particularly for 229E and OC43; however, less information regarding seroprevalence is available for HKU1 and NL63. To measure exposure rates to these four coronavirus strains (229E, HKU1, NL63, and OC43), we devised an immunoassay based on amino- and carboxy-terminally tagged recombinant coronavirus nucleocapsid antigens. Four human and one feline coronavirus antigen were cloned into baculoviruses expressed in insect cells and recovered proteins bound in the solid phase of an enzyme-linked immunosorbent assay-based system. We screened sera from 10 children and 196 adults and established primary cutoff points based on immunoglobulin G (IgG) antibody levels of the predominantly seronegative children. The proportion of seropositive adults for each coronavirus was as follows: 229E, 91.3%; HKU1, 59.2%; NL63, 91.8%; and OC43, 90.8%. No evidence of a significant serological response to the feline coronavirus was observed. Significant associations of coronavirus seropositivity and antibody levels with age, gender, race, socioeconomic status, smoking status, and season of the blood draw were tested with chi-square and regression analyses. The group II coronaviruses (OC43 and HKU1) were significantly associated with race (P<or=0.009 and P<or=0.03, respectively). Elevated OC43 IgG levels were further significantly associated with smoking status (P<or=0.03), as were high NL63 titers with socioeconomic status (P<or=0.04). The high-level immunoreactivity of each coronavirus was significantly associated with the summer season (P<or=0.01 to 0.0001). In summary, high rates of exposure to 229E, NL63, and OC43 and a moderate rate of exposure to HKU1 characterized the seroprevalence among individuals in this population. Demographic factors, such as race, smoking status, and socioeconomic status, may confer an increased risk of susceptibility to these viruses.

Title: Singapore claims first use of antibody test to track coronavirus infections ;Science ;AAAS Content: In what appears to be a first, disease trackers in Singapore have used an experimental antibody test for COVID-19 to confirm that a suspected patient was infected with the coronavirus The patient was one of two people who together formed a missing link between two clusters of cases that each occurred in a Singaporean church Researchers around the world are racing to develop antibody tests, also called serological tests, that can confirm whether someone was infected even after their immune system has cleared the virus that causes COVID-19 The group that developed the test, at Duke-NUS Medical School in Singapore, is among the front-runners, although its assay has to be validated before it is taken into production and deployed widely

Title: Enzyme-linked immunosorbent assay for detection of antibody in volunteers experimentally infected with human coronavirus strain 229 E. Content: An enzyme-linked immunosorbent assay was developed for detecting antibody rises to human coronavirus strain 229E and related strains in paired sera from infected volunteers. There was a close correlation between development of colds infected volunteers. There was a close correlation between development of colds and significant antibody rises detected by the enzyme-linked immunosorbent assay. Furthermore, the assay was more sensitive than a neutralization assay. This enzyme-linked immunosorbent assay is an easy, accurate, and sensitive assay for measuring significant antibody rises to human coronavirus strain 229E group viruses, and it could be useful in the clinical diagnosis of these infections.

Title: SARS-CoV-2 specific antibody responses in COVID-19 patients Content: A new coronavirus, SARS-CoV-2, has recently emerged to cause a human pandemic. Whereas molecular diagnostic tests were rapidly developed, serologic assays are still lacking, yet urgently needed. Validated serologic assays are important for contact tracing, identifying the viral reservoir and epidemiological studies. Here, we developed serological assays for the detection of SARS-CoV-2 neutralizing, spike- and nucleocapsid-specific antibodies. Using serum samples from patients with PCR-confirmed infections of SARS-CoV-2, other coronaviruses, or other respiratory pathogenic infections, we validated and tested various antigens in different in-house and commercial ELISAs. We demonstrate that most PCR-confirmed SARS-CoV-2 infected individuals seroconverted, as revealed by sensitive and specific in-house ELISAs. We found that commercial S1 IgG or IgA ELISAs were of lower specificity while sensitivity varied between the two, with IgA showing higher sensitivity. Overall, the validated assays described here can be instrumental for the detection of SARS-CoV-2-specific antibodies for diagnostic, seroepidemiological and vaccine evaluation studies.

Title: [Seasonal patterns of coronavirus infections in Hamburg]. Content: During a serological surveillance programme between October 1974 and September 1975 in Hamburg, 1624 sera from all age groups of the population were examined for the presence of coronavirus antibodies (OC 43 agent). 61,51% of the sera showed HI-titres between 1:8 and 1:128. The geometric mean antiboty titre (GMT) for the whole group was 1:12,2. The serological seasonal pattern suggests that coronaviruses are circulating the whole year in the urban population with a prevalence in the cold and a decrease in the warm season (June). The highest incidence of seropositive reactions (82,9%) was found in the 15-24 years old individuals (GMT 1:13,8). These data indicate an increased spread of coronaviruses in this segment of the population and a marked susceptibility of the group.

Title: Prevalence of human coronavirus antibody in the population of southern iraq Content: Sera from adults in Southern Iraq were collected during winter and screened by an enzyme–linked immunosorbent assay for the presence of antibodies to the two antigenic groups of human coronaviruses, the 229E and the OC43 groups: 91% of the sera had antibodies to at least one of the groups, whereas 4 and 5% of the sera had antibodies to only the 229E or OC43 groups, respectively. There was significant correlation between the levels of antibody to the 229E and OC43 group coronaviruses in these sera.

Title: Nonspecific inhibitors of coronavirus OC43 haemagglutination in human sera Content: Antibodies against the human coronavirus OC43 in human sera were measured by haemagglutination inhibition (HI), complement fixation (CF), and radial diffusion-haemolysis in gel (HIG) techniques. The apparent HI titres in a fraction of sera with no antibodies detectable by the two other methods were found to be reduced considerably after treating the sera with phospholipase C (PLC). The PLC treatment also reduced the apparent HI titres in some sera containing variable amounts of CF and/or HIG antibodies, but did not affect antibody determinations by the two latter methods. These results suggest that false positive results can be obtained in the OC43 HI test unless the sera are treated with phospholipase C before the assay.

Title: Feline infectious peritonitis: a worldwide serosurvey. Content: Feline sera from 13 countries were assayed for coronavirus antibody, using a heterologous indirect immunofluorescence test. Significantly higher percentages of antibody carriers were obtained during testing randomly collected sera from mature males (greater than 1 year old) than in testing females of the same age. Antibodies were infrequently found in immature cats (less than 6 months old); at 1 year of age or older, a plateau was reached and little change in the percentage of seroconverted animals was observed. Differences were not detected between purebred cats vs mixed-breed cats or household vs stray cats. In animals showing clinical signs of feline infection peritonitis (FIP), antibodies were encountered with higher frequency than in clinically healthy cats. Significant differences in antibody incidence were found between countries, with a range between less than 10% and greater than 50% of seropositive individuals. Antibodies were detected in sera from an isolated cat population (Marion Island) and from wild-caught cheetahs (Acinonyx jubatus). The antibody specificity for FIP virus was confirmed by neutralization tests. The antibody pattern in randomly collected solitary cats, in catteries, and cats with clinical FIP showed characteristic differences in titer and incidence. The implications of these results for the epizootiology of FIP are discussed.

Title: A two year serological surveillance of coronavirus infections in Hamburg Content: The occurrence of OC-43 coronavirus-like infections in the population of Hamburg was determined by a monthly serological survey (hemagglutination inhibition test [HI]) undertaken between October 1974 and October 1976. Studies of 3,016 sera revealed a high incidence of HI antibodies (58.2%). The frequency of seropositive reactions (=1:8) and the geometric mean titer were higher in individuals 15 to 24 years old. Treatment of 50 positive sera (titer 1:16 – 1:64) with receptor destroying enzyme, kaolin and ethacridin for elimination of nonspecific inhibitors did not modify the HI-titer of the investigated sera. The serological seasonal pattern suggests that coronaviruses are circulating the whole year in the urban population with a prevalence in the fall-winter period. Of the 331 paired sera examined, 6.6% presented a four- to eightfold rise in titer within eight weeks.

Title: SEROEPIDEMIOLOGIC SURVEY OF CORONAVIRUS (STRAIN OC 43) RELATED INFECTIONS IN A CHILDREN'S POPULATION Content: Kaye, H. S. (CDC, Atlanta, Ga. 30333), H. B. Marsh and W. R. Dowdle. Sero-epidemiologic survey of coronavirus (strain OC 43) related infections in a children's population. Amer J Epid 94: 43–49, 1971.—Acute and convalescent serum pairs and control sera collected from subjects living in a children's home over a 7-year period (1960–1967) were examined by hemagglutination-inhibition (HI) test with coronavirus strain OC 43. Ninety-three serologic conversions were observed; 44 were associated with reported illnesses and 49 with no reported illnesses. In three distinct outbreaks during the winter and spring quarter of 1960–1961, 1964–1965, and 1966–1967, 67 conversions occurred. Seroconversions to strain OC 43 were associated with as much as 19% of the respiratory diseases in a single season. Over the 7-year period coronavirus strain OC 43 accounted for 3% of the total 1328 respiratory illnesses. Evidence of preexisting antibody was apparent in one-third of the children showing seroconversions. The HI test was more sensitive for serodiagnosis than the complement-fixaticn test. The major presenting complaints of the children with respiratory disease associated with coronavirus strain OC 43 were sore throat, cough and coryza; the predominating symptoms were pharyngitis, coryza, fever and cervical adenitis.

Title: Monoclonal antibody capture enzyme-linked immunosorbent assay for detection of bovine enteric coronavirus. Content: Monoclonal antibodies reactive with three different viral polypeptides were evaluated singly and in combination as the capture antibody(s) in an enzyme-linked immunosorbent assay system for the detection of bovine enteric coronavirus. Similar levels of sensitivity were found for all combinations tested. A sensitive, highly specific, and reproducible assay for the detection of bovine enteric coronavirus was developed, using a mixture of two of these monoclonal antibodies reactive with antigenic components either external or internal to the virion. These monoclonal antibodies were bound indirectly to 96-well plates via rabbit anti-mouse immunoglobulin. After sample application and incubation, virus was detected by using rabbit anti-coronavirus peroxidase conjugate followed by enzyme substrate and chromagen. Fecal samples from a single herd of cows were screened for the presence of coronavirus by this assay. Five percent of clinically normal cows were found to be shedding coronavirus.

Title: Evaluation of nine commercial SARS-CoV-2 immunoassays Content: Due to urgency and demand, numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoassays are rapidly being developed and placed on the market with limited validation on clinical samples. Thorough validation of serological tests are required to facilitate their use in the accurate diagnosis of SARS-CoV-2 infection, confirmation of molecular results, contact tracing, and epidemiological studies. This study evaluated the sensitivity and specificity of nine commercially available serological tests. These included three enzyme-linked immunosorbent assays (ELISAs) and six point-of-care (POC) lateral flow tests. The assays were validated using serum samples from: i) SARS-CoV-2 PCR-positive patients with a documented first day of disease; ii) archived sera obtained from healthy individuals before the emergence of SARS-CoV-2 in China; iii) sera from patients with acute viral respiratory tract infections caused by other coronaviruses or non-coronaviruses; and iv) sera from patients positive for dengue virus, cytomegalovirus and Epstein Barr virus. The results showed 100% specificity for the Wantai SARS-CoV-2 Total Antibody ELISA, 93% for the Euroimmun IgA ELISA, and 96% for the Euroimmun IgG ELISA with sensitivities of 90%, 90%, and 65%, respectively. The overall performance of the POC tests according to manufacturer were in the rank order of AutoBio Diagnostics > Dynamiker Biotechnology = CTK Biotech > Artron Laboratories > Acro Biotech ≥ Hangzhou Alltest Biotech. Overall, these findings will facilitate selection of serological assays for the detection SARS-CoV-2-specific antibodies towards diagnosis as well as sero-epidemiological and vaccine development studies.

Title: Determination of coronavirus 229E antibody by an immune‐adherence hemagglutination method Content: An immune‐adherence hemagglutination (IAHA) method for coronavirus 229E antibody determination has been developed both for diagnosis of recent infections and for detection of long‐past infections. Results have been compared with those obtained by complement fixation (CF), neutralization (Nt), and indirect hemagglutination (IHA) tests. The IAHA method has been shown to be as sensitive as the CF, Nt, and IHA tests in detecting cases of acute 229E infection. However, in a seroepidemiological survey of 343 healthy people of all ages, IAHA detected 229E antibody in 254 individuals (74.0%), Nt in 166 (48.3%), IHA in 89 (25.9%), and CF in 30 (8.7%). A study of the prevalence of coronavirus 229E IAHA antibody in the different age groups has shown that during the second decade of life nearly 100% of the population acquire this type of antibody, whereas only 50% are positive at the end of the first decade. In the older age groups, the high frequency of CF antibody (“marker” of recent infection) indirectly confirms the high rate of 229E reinfections and the nonprotective nature of IAHA antibody. CF titer ⩾ 1:8 in 90% of cases corresponded to IAHA titers ⩾ 1:64. However, sera with IAHA titers of ⩾ 1:128 were often CF‐negative. Recent 229E infections (or reinfections), as determined by the presence of CF antibody, were more frequent in April‐May than in October‐November. Three cases of acute infection showing 229E seroconversion (two adults and one child) were observed during the winter‐spring season. IAHA appears to be the test of choice for seroepidemiological surveys.

Title: Detection of turkey enteric coronavirus by enzyme-linked immunosorbent assay and differentiation from other coronaviruses. Content: A double-antibody ELISA for the detection of coronaviruses in intestinal contents from turkey poults with diarrhea was developed. Antibodies were raised in rabbits and guinea pigs against a Minnesota isolate of turkey enteric coronavirus (TCV) propagated in embryonating turkey eggs and were purified by density-gradient centrifugation. The specificity of antisera was confirmed by hemagglutination-inhibition and immunoelectron microscopy. Absorption of anti-TCV hyperimmune sera with egg extracts or egg ovalbumin and the use of different dilution and blocking buffers influenced the sensitivity and specificity of the ELISA. Reciprocal cross-reactivity was detected among turkey, chicken, bovine, and murine coronaviruses. Antisera to the transmissible gastroenteritis virus of swine, the rabbit enteric coronavirus, or the human coronavirus strain 299E failed to react with TCV. The TCV cross-reacted only moderately with the avian infectious bronchitis virus and the hemagglutinating encephalomyelitis virus of swine. Investigations with samples from 47 commercial turkey flocks in Quebec with episodes of transmissible enteritis revealed that the ELISA was more sensitive than was electron microscopy for detection of TCV.

Title: Evaluation of a computer-assisted, kinetics-based enzyme-linked immunosorbent assay for detection of coronavirus antibodies in cats. Content: A computer-assisted, kinetics-based enzyme-linked immunosorbent assay was adapted for the detection of coronavirus antibodies in feline serum. An alkaline antigen diluent (carbonate-bicarbonate buffer, pH 9.6) used in initial experiments produced diffuse, nonspecific color reactions in both viral and control antigen cuvettes which were correlated, paradoxically, with coronavirus antibody levels in test sera. These interfering reactions were minimized by use of lower-pH antigen diluents such as water and phosphate-buffered saline. Background kinetics-based enzyme-linked immunosorbent assay reactivity directed against a noncoronaviral component of antigen tissue culture fluids could then detected in numerous sera, particularly in samples with lower titers. Much of this reactivity was shown to be associated with bovine gamma globulins in cell culture fluid. It was not serum lot or species specific, since a variety of bovine serum lots as well as individual lots of serum from other mammalian and avian species reacted. Reactivity was markedly reduced when cells for antigen preparation were grown in gamma globulin-free bovine serum. Generation of corrected slope values from the kinetics-based enzyme-linked immunosorbent assay made it possible to correct for residual background reactivity in individual test sera and thus eliminate a potentially major source of false-positive reactions. Collectively, these studies indicated that the control of nonspecific reactivity in feline coronavirus serology is absolutely essential to obtain useful estimates of specific antibody responses.

Title: Two enzyme immunoassays for the detection of antibody to rodent coronaviruses Content: Abstract Two enzyme immunoassays for detection of antibody to rodent coronaviruses were compared. Mouse hepatitis virus (MHV), strain JHM, antigen was in the form of formalin-fixed, infected 17 C1 1 cells. This antigen detected antibody to the homologous strain of MHV as well as to two heterologous MHV strains and a serologically related rat coronavirus, sialodacryodenititis virus. Antibody titers in assays using horseradish peroxidase (HRP)-conjugated or ureiase-conjugated anti-mouse IgG were substantially higher than in an indirect immunofluorescence assay. The ureiase assay was somewhat more sensitive than the HRP assay. MHV-JHM antigen was stable under a variety of storage conditions for at least two months.

Title: Evaluation of commercial and automated SARS-CoV-2 IgG and IgA ELISAs using coronavirus disease (COVID-19) patient samples Content: Antibody-screening methods to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) need to be validated. We evaluated SARS-CoV-2 IgG and IgA ELISAs in conjunction with the EUROLabworkstation (Euroimmun, Lübeck, Germany). Overall specificities were 91.9% and 73.0% for IgG and IgA ELISAs, respectively. Of 39 coronavirus disease patients, 13 were IgG and IgA positive and 11 IgA alone at sampling. IgGs and IgAs were respectively detected at a median of 12 and 11 days after symptom onset.

Title: OC43 strain‐related coronavirus antibodies in different age groups Content: Serum antibodies against human coronavirus OC43 in different age groups were measured by complement fixation (CF), haemagglutination inhibition (HI), radial diffusion haemolysis‐in‐gel (HIG), and solid‐phase radioimmunoassay (RIA) methods. Antigen grown in suckling mouse brain was used in all tests. Results obtained by the CF and HIG tests, and the RIA, were in good agreement with regard to the presence or absence of antibodies. Similar results were also obtained with the HI test if nonspecific haemagglutination inhibitors were first removed by treatment with phospholipase C and only titers of 1:20 or greater were considered positive. Children 6–23 months of age (n = 45) were without measurable coronavirus antibodies in all four assays. A rapid increase in the prevalence of antibodies then occurred in subsequent age groups, and practically all persons 6 years of age or older were found to have OC43 antibodies as measured by the HIG test or the RIA. The mean antibody levels determined by these two methods continued to increase, however, up to the age group of 10–14 years. This increase in antibody levels after the initial antibody incidence plateau may be due to boosting effects caused by related coronavirus strains, since OC43 antigens are known to cross‐react with antibodies induced by other human coronaviruses. Taken together, these data suggest that OC43 virus, or an antigenically related coronavirus strain, is very common in Finland.

Title: Development and application of an enzyme immunoassay for coronavirus OC43 antibody in acute respiratory illness. Content: Study of coronavirus OC43 infections has been limited because of the lack of sensitive cell culture systems and serologic assays. To improve this circumstance, we developed an indirect enzyme immunoassay (EIA) to detect serum antibody to OC43. Antigen (100 ng) prepared by polyethylene glycol precipitation provided optimal results without a postcoat procedure. Evaluation of intraplate variation indicated that a > or = 2.5-fold increase in serum titer was significant. Sixteen of 18 (89%) paired serum samples with previously identified, reproducible increases in the level of hemagglutination inhibition (HAI) antibody to OC43 also showed significant increases as detected by EIA. Specificity for the EIA was established with paired sera obtained from persons given influenza immunizations or experiencing a respiratory infection. No rise in antibody titers occurred among 33 persons with documented coronavirus 229E infection. EIA was then performed on each of 419 paired serum samples from ambulatory chronic obstructive pulmonary disease patients and healthy older adults, from asthmatic adults presenting for emergency room treatment, and from persons hospitalized with acute respiratory symptoms. Twenty-three antibody rises to OC43 were detected; only nine of these were detected by the HAI test, and the HAI test did not detect any increases in antibody titers that were not detected by EIA. Nineteen of 25 coronavirus OC43 infections for which a month of infection could be assigned occurred between November and February. Overall, 4.4% of acute respiratory illnesses in the studied populations were associated with a coronavirus OC43 infection.

Title: Serologic evidence of coronavirus infection in New York and New England dairy cattle with winter dysentery. Content: Acute and convalescent sera were collected from 8 dairy herds with classic clinical features of winter dysentery. An enzyme-linked immunosorbent assay was used to measure coronavirus antibody titers, employing calf diarrhea coronavirus as antigen. Twenty-two of the 35 animals tested (63%) showed a greater than or equal to 4-fold seroconversion. Adult cattle in all 8 herds seroconverted. These findings complement previously reported immunoperoxidase and electron microscopic evidence, suggesting an etiologic role for an enteric coronavirus in this disease.

Title: Coronavirus infections of man associated with diseases other than the common cold Content: About 14,000 paired sera, from patients with various types of acute infectious diseases with suspected viral origin, were screened by complement fixation against a wide set of viral antigens, including coronavirus OC43. A significant change in OC43 antibodies was recorded in 33 cases and a constant high titre, defined as a titre occurring in the respective age group in less than 1 % of all sera examined, was found in 45 cases. On the basis of careful retrospective analysis of hospital case records it was concluded that in 28 cases with an increase of OC43 antibody litres, and in two with titre decrease, a disease could be associated with an acute coronavirus infection. In 16 cases the disease was dominated by respiratory symptoms. Eight of these patients, four children and four adults, had pneumonia. Three of the eight pneumonia patients had, however, another concomitant infection, too. Four patients had neurological symptoms, one had severe perimyocarditis, and in five cases fever was the only symptom recorded. Among the patients with a statistically significant high titre of OC43 antibodies, there were 14 cases where a suggestive association with a disease could be envisaged on the basis of hospital records. Five of these patients had pneumonia. These results suggest that human coronaviruses, so far considered only as one group of causative agents of the common cold, may also be associated with other and more severe diseases in all age groups.

Title: Seroepidemiology of group I human coronaviruses in children Content: BACKGROUND: Recently, several new human coronaviruses have been identified. OBJECTIVES: To define the seroepidemiology of group I human coronaviruses STUDY DESIGN: A recombinant protein enzyme linked immunosorbent assay (ELISA) based on portions of the nucleocapsid protein of group I human coronaviruses was developed and was used to screen serum from 243 children and young adults. RESULTS: For HCoV-229E, the percentages of seropositive individuals were 57.1% for infants < 2 months old; 38.9% for infants 2–3 months old; 4.7% for infants 4–5 months old; 42.9–50.0% for infants 6–12 months old; and 34.8–62.5% for individuals 1–20 years old. For HCoV-NL63, the percentages of seropositive individuals were 45.2% for infants < 2 months old; 11.1% for infants 2–3 months old; 4.7% for infants 4–5 months old; 28.6–40.0% for infants 6–12 months old; and 25.0–70.3% for individuals 1–20 years old. CONCLUSIONS: Infection with these viruses is common in childhood though the prevalence of these viruses may vary from year to year.

Title: Analysis of SARS-CoV-2 Antibodies in COVID-19 Convalescent Plasma using a Coronavirus Antigen Microarray Content: The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates complete discrimination of these two groups. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.

Title: Cats, coronaviruses and coronavirus antibody tests Content: Feline infectious peritonitis and other coronavirus infections of cats are briefly reviewed. Interpretation and applications of feline coronavirus antibody tests are described, and general recommendations are provided for practitioners. Some of the major unresolved questions regarding coronavirus infections of cats are delineated.

Title: Testing For SARS-CoV-2: The Day the World Turned its Attention to the Clinical Laboratory Content: In the last few months, an unprecedented number of laboratory tests for coronavirus disease 2019 (COVID-19) have been developed at a remarkable speed. With the rapid adoption of these tests into clinical practice, combined with the widespread publicity they received, questions arose related to the different types of tests, their utility, performance, and regulatory approval status. The aim of this publication is to provide a general landscape of laboratory testing for COVID-19 and offer a historical and regulatory perspective associated with them. Specifically, we aim to elaborate on the regulatory complexities of diagnostic testing in the United States and its implications to the present outbreak, as well as provide a synopsis of laboratory tests that have been developed for COVID-19. We will first address the detection of severe acute respiratory syndrome-coronavirus 2 directly by either nucleic acid amplification tests or by the detection of the viral protein for active infections. Subsequently, we will provide an overview of serological tests that can aid not only in diagnosis but additionally help to identify prior infections and potential immunity.

Title: Performance of Severe Acute Respiratory Syndrome Coronavirus 2 Antibody Assays in Different Stages of Infection: Comparison of Commercial Enzyme-Linked Immunosorbent Assays and Rapid Tests Content: We comparatively assessed sensitivities and specificities of 4 commercial enzyme-linked immunosorbent assays (ELISAs) and 2 rapid tests in 77 patients with polymerase chain reaction-confirmed severe acute respiratory syndrome coronavirus 2 infection, grouped by interval since symptom onset. Although test sensitivities were low (<40%) within the first 5 days after disease onset, immunoglobulin (Ig) M, IgA, and total antibody ELISAs increased in sensitivity to >80% between days 6 and 10 after symptom onset. The evaluated tests (including IgG and rapid tests) provided positive results in all patients at or after the 11th day after onset of disease. The specificities of the ELISAs were 83% (IgA), 98% (IgG), and 97% (IgM and total antibody).

Title: Specific serology for emerging human coronaviruses by protein microarray. Content: We present a serological assay for the specific detection of IgM and IgG antibodies against the emerging human coronavirus hCoV-EMC and the SARS-CoV based on protein microarray technology. The assay uses the S1 receptor-binding subunit of the spike protein of hCoV-EMC and SARS-CoV as antigens. The assay has been validated extensively using putative cross-reacting sera of patient cohorts exposed to the four common hCoVs and sera from convalescent patients infected with hCoV-EMC or SARS-CoV.

Title: Evolving status of the 2019 novel coronavirus infection: Proposal of conventional serologic assays for disease diagnosis and infection monitoring Content: The novel coronavirus (nCoV-2019) outbreak in Wuhan, China has spread rapidly nationwide, with some cases occurring in other parts of the world. Although most patients present with mild febrile illness with patchy pulmonary inflammation, a significant portion develop severe acute respiratory distress syndrome (ARDS), with a current case fatality of 2.3-3%. Diagnosis is based on clinical history and laboratory and chest radiographic findings, but confirmation currently relies on nucleic acid-based assays. The latter are playing an important role in facilitating patient isolation, treatment and assessment of infectious activities. However, due to their limited capacity to handle an epidemic of the current scale and insufficient supply of assay kits, only a portion of suspected cases can be tested, leading to incompleteness and inaccuracy in updating new cases, as well as delayed diagnosis. Furthermore, there has not been enough time to assess specificity and sensitivity. Conventional serological assays, such as enzyme-linked immunoassay (ELISA) for specific IgM and IgG antibodies, should offer a high-throughput alternative, which allows for uniform tests for all suspected patients, and can facilitate more complete identification of infected cases and avoidance of unnecessary cross infection among unselected patients. This article is protected by copyright. All rights reserved.

Title: Development and applications of a bovine coronavirus antigen detection enzyme-linked immunosorbent assay. Content: We developed a monoclonal antibody-based, antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) for bovine coronavirus. We compared the ELISA with electron microscopy and the hemagglutination test and found a close correlation between them. The sensitivity of the ELISA was 10(4) bovine coronavirus particles per ml of 10% fecal suspension. Compared with electron microscopy, bovine coronavirus ELISA had 96% specificity.

Title: Detection by radioimmunoassay and enzyme-linked immunosorbent assay of coronavirus antibodies in bovine serum and lacteal secretions. Content: The sensitivity of a radioimmunoassay (RIA), an enzyme-linked immunosorbent assay (ELISA), and a serum neutralization assay (SN) for detecting antibodies to bovine coronavirus in serum and colostrum were compared. Although there proved to be a good correlation among all three assays (r = 0.915 and 0.964 for RIA with SN and ELISA, respectively), RIA and ELISA proved to be at least 10 times more sensitive than neutralization tests. By using these techniques, it was possible to detect a time-dependent decrease in antibody levels in bovine colostrum after parturition. Using ELISA, we demonstrated that 12 of 12 herds in Saskatchewan, and 109 of 110 animals tested, and antibody to bovine coronavirus. There was no elevated antibody response in serum or lacteal secretions of cows vaccinated once or twice with a commercially available modified live rota-coronavirus vaccine. In addition to being more sensitive than SN, ELISA and RIA proved to have other advantages for measuring antibody levels to bovine coronavirus and therefore warrant wider use as tools in diagnostic virology.

Title: Coronavirus antibodies in sera from patients with multiple sclerosis and matched controls. Content: Sera from patients with multiple sclerosis and carefully matched controls were tested for antibodies to three strains of coronavirus. There was no significant difference in the levels of antibody in the patients vs the controls. We conclude that unless the strains of coronaviruses recently reported to have been isolated from patients with multiple sclerosis express important serological differences from those used in these studies, coronaviruses are not associated with the cause of multiple sclerosis.

Title: The spectrum of severe acute respiratory syndrome-associated coronavirus infection. Content: BACKGROUND Whether subclinical or atypical presentations of severe acute respiratory syndrome (SARS) occur and whether clinical judgment is accurate in detecting SARS are unknown. OBJECTIVES To describe the spectrum of SARS coronavirus infection in a large outbreak and to compare diagnoses based on clinical judgment with the SARS coronavirus test. DESIGN Secondary analysis of prospectively collected clinical data and archived serum. SETTING A SARS screening clinic of a university hospital in the New Territories of Hong Kong. PATIENTS 1221 patients attending the clinic between 12 March 2003 and 12 May 2003. MEASUREMENTS SARS coronavirus serology. RESULTS 145 of 553 (26%) patients had serologic evidence of SARS coronavirus infection. Of 910 patients who were managed without hospitalization, only 6 had serologic evidence of SARS. Five of the six patients had normal chest radiographs, and four had symptoms such as myalgia, chills, coughing, and feeling feverish. With the SARS coronavirus serologic test as the gold standard, the clinical diagnosis of probable SARS at hospitalization had a sensitivity of 0.96 (95% CI, 0.91 to 0.98) and a specificity of 0.96 (CI, 0.92 to 0.97). LIMITATIONS Follow-up serologic samples were not obtained from almost half of the patients because they declined further testing. Some people living in the community who were infected but who had minor or no symptoms might not have visited the clinic. CONCLUSIONS There is little evidence of widespread subclinical or mild forms of SARS coronavirus infection. Clinical diagnoses during the outbreak were reasonable and resulted in appropriate triaging.

Title: The Tecumseh Study of Respiratory Illness. VI. Frequency of and Relationship between Outbreaks of Coronavims Infection Content: Specimens of blood collected in Tecumseh, Michigan over a four-year period were studied for rise in antibody titer against coronavirus OC43. Peaks of infection were found in the winter and spring of 1966, 1968, and 1969; at other times, infections occurred sporadically. All age groups were involved, especially the very young. Rises in titer by CF and by HAI tests frequently did not occur together in the same individual. Agreement between the two tests was better in 1966 and 1969 than in the other years. A portion of the paired specimens showing rises in CF and/ or HAI titer was tested by neutralization. Rises in neutralizing antibody were usually found in pairs collected in 1966 and 1969 but not in those collected in 1967 and 1968. The infecting viruses in 1966 and 1969 thus appeared more closely related to OC43 than did those in 1967 and 1968.

Title: First external quality assurance of antibody diagnostic for SARS-new coronavirus Content: Abstract To confirm an infection with the new coronavirus (SARS-CoV) causing the severe acute respiratory syndrome (SARS) diagnostic assays for detection of SARS-CoV specific antibody are necessary. To evaluate the diagnostic performance of laboratories an external quality assurance (EQA) study was performed in 2004. Participating laboratories (9/20) correctly detected anti-SARS antibodies in serum samples without false positive results in an immunofluorescence assay. In contrast, only 4/13 laboratories detected most of the anti-SARS antibody positive samples without false positive results using enzyme immunoassays (EIA) and/or immunoblot. The overall results clearly demonstrate that serological diagnosis of SARS-CoV remains at an early stage of development, with further technical improvements required, particularly with respect to the use of SARS specific EIAs.

Title: [Serological diagnostic studies of the occurrence of coronavirus infections in cattle with respiratory diseases]. Content: Of paired serum samples of 196 cattle with respiratory disease from 37 herds which were tested for hemagglutination inhibiting antibodies against coronavirus (BCV) 185 of the first specimens and 190 of the second ones gave positive results. This difference was due to five animals of five different farms showing seroconversions between the time of the blood collections. A significant rise in antibody titer (no less than 4-fold) was evident in 28 cattle from 3 of the above mentioned and 12 other farms. Altogether serological indication of acute coronavirus infections were gained in 33 (16.8%) of animals with respiratory diseases which derived from 17 of 37 herds. Most of these infections (about 35%) occurred in animals older than 6 months while calves up to 3 or 6 months were only affected in a ratio of ca. 10%, respectively.

Title: Enquête sérologique par immunofluorescence indirecte, sur les infections à coronavirus OC43 Content: Summary Four-hundred seventy-two sera were examined by the indirect immunofluorescence test against Coronavirus strain OC43 adapted to the HRT18 cell culture. Sera were from children and adults hospitalized over a 4-year period, from 1979 to 1982, at the CHRU of Clermont-Ferrand (France) for different clinical reasons. Of the 446 sera tested, 61 % showed antibodies against OC43; 90 % of adult sera were positive. Coronavirus infections are very common in France. Immunity appears early in life, and infection is frequent in children from 12–36 months of age.

Title: Evaluation of the EUROIMMUN Anti-SARS-CoV-2 ELISA Assay for detection of IgA and IgG antibodies Content: As the Coronavirus 2019 (COVID-19) pandemic evolves, the development of immunoassays to help determine exposure and potentially predict immunity has become a pressing priority. In this report we present the performance of the EUROIMMUN enzyme-linked immunosorbent assay (ELISA) for semi-quantitative detection of IgA and IgG antibodies in serum and plasma samples using recombinant S1 domain of the SARS-CoV-2 spike protein as antigen. Specimens from patients, with and without COVID-19 infection, were tested at the University of Chicago Clinical Microbiology and Immunology Laboratory. Of 57 samples from COVID-19 PCR-negative patients, including 28 samples positive for common human coronavirus strains, 53 tested negative and 4 tested positive for IgA (93.0% agreement) while 56 tested negative and 1 tested positive for IgG (98.2% agreement). For COVID-19 PCR-positive patients, 29 of 30 (96.7%) samples collected ≥3 days after positive PCR were positive for IgA, and 28 of 28 samples collected ≥4 days after positive PCR were positive for IgG. The EUROIMMUN Anti-SARS-CoV-2 ELISA Assay demonstrates excellent sensitivity for detection of IgA and IgG antibodies from samples collected ≥3 days and ≥4 days, respectively, after COVID-19 diagnosis by PCR. This assay did not demonstrate cross reaction in any of the 28 samples from patients with common human coronaviruses, including types HKU1, NL63, CV229E, and OC43.

Title: Dynamic profile for the detection of anti-SARS-CoV-2 antibodies using four immunochromatographic assays Content: In order to fight the SARS-CoV-2 pandemic infection, there is a growing need and demand for diagnostic tools that are complementary and different from the RT-PCR currently in use. Multiple serological tests are or will be very soon available but need to be evaluated and validated. We have thus tested 4 immunochromatographic tests for the detection of antibodies to SARS-CoV-2. In addition, we assessed the kinetics of antibody appearance using these assays in 22 patients after they were tested positive by RT-PCR. We observed great heterogeneity in antibody detection post-symptom onset. The median antibody detection time was between 8 and 10 days according to the manufacturers. All the tests showed a sensitivity of 60 to 80% on day 10 and 100% on day 15. In addition, a single cross-reaction was observed with other human coronavirus infections. Thus, immunochromatographic tests for the detection of anti-SARS-CoV-2 antibodies may have their place for the diagnostic panel of COVID-19.

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

Title: Evaluating the serological status of COVID-19 patients using an indirect immunofluorescent assay, France. Content: An indirect immunofluorescent assay was developed in order to assess the serological status of 888 RT-PCR-confirmed COVID-19 patients (1,302 serum samples) and controls in Marseille, France. Incorporating an inactivated clinical SARS CoV-2 isolate as the antigen, the specificity of the assay was measured as 100% for IgA titre [≥] 1:200; 98.6% for IgM titre [≥] 1:200; and 96.3% for IgG titre [≥] 1:100 after testing a series of negative controls as well as 150 serums collected from patients with non-SARS-CoV-2 Coronavirus infection, non-Coronavirus pneumonia and infections known to elicit false-positive serology. Seroprevalence was then measured at 3% before a five-day evolution up to 47% after more than 15 days of evolution. We observed that the seroprevalence as well as the titre of specific antibodies were both significantly higher in patients with a poor clinical outcome than in patients with a favourable evolution. These data, which have to be integrated into the ongoing understanding of the immunological phase of the infection, suggest that serotherapy may not be a therapeutic option in patients with severe COVID-19 infection. The IFA assay reported here is useful for monitoring SARS-CoV-2 exposure at the individual and population levels.

Title: [Development of a SPA-ELISA method for detecting anti-coronavirus IgG antibodies in serum samples from fulvous fruit bats]. Content: OBJECTIVE To develop an simple and sensitive method for detecting anti-coronavirus IgG antibodies in bat sera based on enzyme-linked immunosorbent assay (ELISA). METHODS A commercial ELISA kit for detecting SARS-CoV antibody was modified for detecting coronavirus antibodies in bat serum samples. The second antibody in the kit was replaced with horseradish peroxidase-conjugated protein-A (HRP-SPA) based on the characteristics of binding between Staphylococcus aureus protein A (SPA) and mammal IgG Fc fragment. The sera of 55 fulvous fruit bats (Rousettus dasymallus) were tested using the SPA-ELISA. RESULTS The test results of the positive and negative controls in the kit and the serum samples from convalescent ;patient were consistent with expectation. Coronavirus antibody was detected in 2 out of the 55 bat serum samples. Serum neutralization test confirmed the validity of the SPA-ELISA method. CONCLUSION This SPA-ELISA method is applicable for detecting coronavirus antibody in bat sera.

Title: Serological differentiation between COVID-19 and SARS infections Content: In response to the coronavirus disease 2019 (COVID-19) outbreak, caused by the SARS-CoV-2 virus, multiple diagnostic tests are required globally for acute disease diagnosis, contact tracing, monitoring of asymptomatic infection rates and assessing herd immunity. While PCR remains the frontline test of choice in the acute diagnostic setting, serological tests are urgently needed to fulfil the other requirements. Unlike PCR tests which are highly specific for each virus, cross-reactivity could potentially be a major challenge for COVID-19 antibody tests considering there are six other coronaviruses known to infect humans. Among the human pathogens, SARS-CoV is genetically most related to SARS-CoV-2 sharing approximately 80% sequence identity and both belong to the species SARS related coronavirus (SARSr-CoV) in the genus Betacoronavirus of family Coronaviridae. In this study, we developed and compared the performance of four different serological tests to comprehensively assess the cross-reactivity between COVID-19 and SARS patient sera. Our results indicate that there is a significant cross-reactivity when N protein of either SARS-CoV or SARS-CoV-2 is used. The S1 or RBD derived the spike (S) protein offers better specificity. Amongst the different platforms, capture ELISA performed best. Finally, we found that SARS survivors all have significant level of antibodies remaining in their blood 17 years after infection. We discovered that anti-N antibodies waned more than anti-RBD antibodies, and the latter is known to play a more important role in providing protective immunity.

Title: Serological differentiation between COVID-19 and SARS infections. Content: In response to the coronavirus disease 2019 (COVID-19) outbreak, caused by the SARS-CoV-2 virus, multiple diagnostic tests are required globally for acute disease diagnosis, contact tracing, monitoring of asymptomatic infection rates and assessing herd immunity. While PCR remains the frontline test of choice in the acute diagnostic setting, serological tests are urgently needed to fulfil the other requirements. Unlike PCR tests which are highly specific for each virus, cross-reactivity could potentially be a major challenge for COVID-19 antibody tests considering there are six other coronaviruses known to infect humans. Among the human pathogens, SARS-CoV is genetically most related to SARS-CoV-2 sharing approximately 80% sequence identity and both belong to the species SARS related coronavirus (SARSr-CoV) in the genus Betacoronavirus of family Coronaviridae. In this study, we developed and compared the performance of four different serological tests to comprehensively assess the cross-reactivity between COVID-19 and SARS patient sera. Our results indicate that there is a significant cross-reactivity when N protein of either SARS-CoV or SARS-CoV-2 is used. The S1 or RBD derived the spike (S) protein offers better specificity. Amongst the different platforms, capture ELISA performed best. Finally, we found that SARS survivors all have significant level of antibodies remaining in their blood 17 years after infection. We discovered that anti-N antibodies waned more than anti-RBD antibodies, and the latter is known to play a more important role in providing protective immunity.

Title: Prevalence of subclinical infection by the SARS coronavirus among general practitioners in Hong Kong. Content: Eight general practitioners had severe acute respiratory distress syndrome (SARS) in Hong Kong during the epidemic, and others may have been infected by the SARS coronavirus without developing the full syndrome. We conducted a serological and questionnaire survey to determine the prevalence of subclinical infection by SARS coronavirus among general practitioners in Hong Kong. Participants had to be doctors actively practising in family medicine and who did not have SARS. Approximately 3200 general practitioners were invited to participate and the results of 574 were eligible for analysis. 29 samples were tested positive by enzyme-linked immunosorbent assay, but all these samples had titre < 25 by immunoflorescence assay. The prevalence for seropositivity was thus 0% (95% CI, 0.0%-0.6%). This finding documents the lack of subclinical infection by SARS coronavirus in an at-risk group in the community.

Title: Efficacy of Serology Testing in Predicting Reinfection in Patients with SARS-CoV-2. Content: In many parts of the United States, SARS-CoV-2 cases have reached peak infection rates, prompting administrators to create protocols to resume elective cases. As elective procedures and surgeries get scheduled, ASCs must implement some form of widespread testing in order to ensure the safety of both the ASC staff as well as the patients being seen. The CDC recently announced the approval of new serological testing for SARS-CoV-2, a test that can indicate the presence of IgM and IgG antibodies in the serum against viral particles. However, the possibility for reinfection raises questions about the utility of this new serological test, as the presence of IgG may not correspond to long-term immunity. The coronavirus has been known to form escape mutations, which may correspond to reduction in immunoglobulin binding capacity. Patients who develop more robust immune responses with formation of memory CD8+ T-cells and helper CD4+ T-cells will be the most equipped if exposed to the virus, but unfortunately the serology test will not help us in distinguishing those individuals. Given the inherent disadvantages of serological testing, antibody testing alone should not be used when deciding patient care and should be combined with PCR testing.

Title: Efficacy of Serology Testing in Predicting Reinfection in Patients with SARS-CoV-2 Content: In many parts of the United States, SARS-CoV-2 cases have reached peak infection rates, prompting administrators to create protocols to resume elective cases. As elective procedures and surgeries get scheduled, ASCs must implement some form of widespread testing in order to ensure the safety of both the ASC staff as well as the patients being seen. The CDC recently announced the approval of new serological testing for SARS-CoV-2, a test that can indicate the presence of IgM and IgG antibodies in the serum against viral particles. However, the possibility for reinfection raises questions about the utility of this new serological test, as the presence of IgG may not correspond to long-term immunity. The coronavirus has been known to form escape mutations, which may correspond to reduction in immunoglobulin binding capacity. Patients who develop more robust immune responses with formation of memory CD8+ T-cells and helper CD4+ T-cells will be the most equipped if exposed to the virus, but unfortunately the serology test will not help us in distinguishing those individuals. Given the inherent disadvantages of serological testing, antibody testing alone should not be used when deciding patient care and should be combined with PCR testing.

Title: Preliminary Results of Seroprevalence of SARS-CoV-2 at Community Clinics in Tokyo Content: Serological evaluation with SARS-CoV-2 specific IgG antibody will be an alternative way to know the pandemic of novel coronavirus disease (COVID-19) if the capacity for diagnostic PCR test is limited. The point-of-care test to detect SARS-CoV-2 specific IgG antibody in peripheral blood (n =202) was performed in two community clinics in Tokyo, Japan. The overall positive rate of SRAS-CoV-2 IgG antibody was 5.9% (95% confidence interval[CI]: 3.1-10.1). Higher rate was observed for healthcare workers (n =55, 9.1 [3.0-20.0]). The limitation on antibody tests includes low sensitivity and potent cross-reactivity with the previous coronavirus. Robust healthcare policy to efficiently monitor COVID-19 spread is warranted in Tokyo.

Title: SARS-CoV-2 antibody testing-questions to be asked Content: Severe acute respiratory syndrome coronavirus 2 infection and development of coronavirus disease 2019 presents a major health care challenge of global dimensions. Laboratory diagnostics of infected patients, and the assessment of immunity against severe acute respiratory syndrome coronavirus 2, presents a major cornerstone in handling the pandemic. Currently, there is an increase in demand for antibody testing and a large number of tests are already marketed or are in the late stage of development. However, the interpretation of test results depends on many variables and factors, including sensitivity, specificity, potential cross-reactivity and cross-protectivity, the diagnostic value of antibodies of different isotypes, and the use of antibody testing in identification of acutely ill patients or in epidemiological settings. In this article, the recently established COVID-19 Task Force of the German Society for Clinical Chemistry and Laboratory Medicine (DGKL) addresses these issues on the basis of currently available data sets in this rapidly moving field.

Title: Identification of antibody against porcine coronavirus in human milk. Content: A total of 239 human milk samples collected from mothers in Chiba City, Japan during 1994-1997 were tested for the presence of anti-coronavirus antibody. Interestingly, twelve human milk samples were positive for IgA against porcine transmissible gastroenteritis coronavirus, and this represented 5%. These findings provided evidence that the interspecies transmission of coronavirus between human and porcine might occur in nature. This report is noteworthy because it is the first, to the best of our knowledge, demonstrating the presence of antibody against porcine transmissible gastroenteritis coronavirus in human milk.

Title: Antibodies to coronaviruses are higher in older compared with younger adults and binding antibodies are more sensitive than neutralizing antibodies in identifying coronavirus‐associated illnesses Content: Human coronaviruses (HCoV) are common causes of respiratory illnesses (RI) despite preexisting humoral immunity. Sera were obtained near the onset of RI and 3 to 4 weeks later as part of a prospective study of 200 subjects evaluated for RI from 2009 to 2013. Antibodies against common HCoV strains were measured by enzyme‐linked immunosorbent assay and neutralization assay comparing older adults with cardiopulmonary diseases (99 subjects) to younger, healthy adults (101 subjects). Virus shedding was detected in respiratory secretions by polymerase chain reaction. Of 43 HCoV‐associated illnesses, 15 (35%) occurred in 14 older adults (aged ≥60 years) and 28 (65%) in 28 younger adults (aged 21‐40 years). Binding and neutralizing antibodies were higher in older adults. Only 16 (35.7%) of RI with increases in binding antibodies also had increases in neutralizing antibodies to HCoV. Increases in binding antibodies with RI were more frequent than increased neutralizing antibodies and virus shedding, and more frequent in younger compared to older adults. Functional neutralizing antibodies were not stimulated as often as binding antibodies, explaining in part a susceptibility to reinfection with HCoV. Monitoring binding antibodies may be more sensitive for the serologic detection of HCoV infections.

Title: Evaluation of the performance of SARS-CoV-2 serological tools and their positioning in COVID-19 diagnostic strategies Content: Rapid and accurate diagnosis is crucial for successful outbreak containment. During the current coronavirus disease 2019 (COVID-19) public health emergency, the gold standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection diagnosis is the detection of viral RNA by reverse transcription (RT)-PCR. Additional diagnostic methods enabling the detection of current or past SARS-CoV-2 infection would be highly beneficial to ensure the timely diagnosis of all infected and recovered patients. Here, we investigated several serological tools, i.e., two immunochromatographic lateral flow assays (LFA-1 (Biosynex COVID-19 BSS) and LFA-2 (COVID-19 Sign IgM/IgG)) and two enzyme-linked immunosorbent assays (ELISAs) detecting IgA (ELISA-1 Euroimmun), IgM (ELISA-2 EDI) and/or IgG (ELISA-1 and ELISA-2) based on well-characterized panels of serum samples from patients and healthcare workers with PCR-confirmed COVID-19 and from SARS-CoV-2-negative patients. A total of 272 serum samples were used, including 62 serum samples from hospitalized patients (panel 1 and panel 3), 143 serum samples from healthcare workers (panel 2) diagnosed with COVID-19 and 67 serum samples from negative controls. Diagnostic performances of each assay were assessed according to days after symptom onset (dso) and the antigenic format used by manufacturers. We found overall sensitivities ranging from 69% to 93% on panels 1 and 2 and specificities ranging from 83% to 98%. The clinical sensitivity varied greatly according to the panel tested and the dso. The assays we tested showed poor mutual agreement. A thorough selection of serological assays for the detection of ongoing or past infections is advisable.

Title: Detection of specific antibodies to severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein for serodiagnosis of SARS coronavirus pneumonia. Content: We report the evaluation of recombinant severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) nucleocapsid protein enzyme-linked immunosorbent assay (ELISA)-based antibody tests for serodiagnosis of SARS-CoV pneumonia and compare the sensitivities and specificities of this ELISA for detection of immunoglobulin G (IgG), IgM, IgA, and their combinations with serum samples from 149 healthy blood donors who donated blood 3 years ago as controls and 106 SARS-CoV pneumonia patients in Hong Kong. The specificities of the ELISA for IgG, IgM, and IgA detection were 95.3, 96.6, and 96.6%, respectively, with corresponding sensitivities of 94.3, 59.4, and 60.4%, respectively. The present ELISA appears to be a sensitive test for serodiagnosis of SARS-CoV pneumonia, is much more economical and less labor-intensive than the indirect immunofluorescence assay, and does not require cultivation of SARS-CoV.

Title: An enzyme-linked immunosorbent assay using canine coronavirus-infected CRFK cells as antigen for detection of anti-coronavirus antibody in cat Content: Abstract From the reasons that canine coronavirus (CCV) grows more efficiently than feline coronavirus in a cell culture and they are mutually related in their antigenicities, an enzyme-linked immunosorbent assay (ELISA) using CCV-infected feline kidney (CRFK) cells as substrate antigens was developed for detection of anti-coronavirus antibodies in cats. It was indispensable for generating coronavirus-specific ELISA antibody activities that the sample was applied to the mock-infected, normal CRFK cells in parallel with the CCV-infected cells and then the optical density values given by the mock-infected cell antigen were subtracted from those given by the virus-infected cell antigen. On the basis of ELISA antibody titers obtained in sera from the cats experimentally infected with CCV and from the spontaneous feline infectious peritonitis (FIP) cases, the ELISA described in the present study was found to be applicable as a simple and easy serologic test which was able to detect anti-coronavirus antibodies as efficiently as the indirect immunofluorescence assay with homologous FIP virus.

Title: Antigenic assessment of coronaviruses isolated from patients with multiple sclerosis. Content: Many studies have either supported or discounted the role of coronaviruses as etiologic agents in multiple sclerosis (MS). Two new approaches were applied to investigate this controversy. First, monoclonal antibodies specific for either murine coronaviruses (mouse hepatitis viruses) or human coronaviruses were used to characterize the antigenic features of MS-derived coronaviruses SK and SD. Both isolates were found to have a mouse hepatitis virus-type profile. Second, serum and cerebrospinal fluid antibodies to different coronaviruses, including SD, were measured in MS and control groups. No significant difference in antibody level to coronaviruses was found between MS and control samples. The results of these antigenic studies do not support a specific association between MS and coronaviruses.

Title: Double-antigen sandwich ELISA for detection of antibodies to SARS-associated coronavirus in human serum Content: The study presented here was conducted to evaluate the performance of a double-antigen sandwich ELISA to detect antibodies in human serum against the coronavirus associated with severe acute respiratory syndrome (SARS). A recombinant partial nucleocapsid protein of SARS-associated coronavirus was used as a serodiagnostic antigen in the ELISA. A total of 2892 clinical serum samples were tested with the ELISA kit, which positively identified 25 of 35 (71.4%) samples of patients with confirmed SARS infection, 286 of 407 (70%) samples of patients suspected of having SARS, 229 of 302 (75.8%) samples of convalescent SARS patients, and 0 of 544 samples obtained from healthcare workers; only 1 of 1604 clinical samples obtained from patients with other diseases demonstrated a weakly positive result. These results indicate that the double-antigen sandwich ELISA is an effective screening method for the serodiagnosis of SARS-associated coronavirus.

Title: Serology characteristics of SARS-CoV-2 infection since exposure and post symptom onset. Content: BACKGROUND Timely diagnosis of SARS-CoV-2 infection is a prerequisite for treatment and prevention. The serology characteristics and complement diagnosis value of the antibody test to RNA test need to be demonstrated. METHOD Serial sera of 80 patients with PCR-confirmed COVID-19 were collected at the First Affiliated Hospital of Zhejiang University, China. Total antibody (Ab), IgM and IgG antibodies against SARS-CoV-2 were detected, and the antibody dynamics during the infection were described. RESULTS The seroconversion rates for Ab, IgM and IgG were 98.8%, 93.8% and 93.8%, respectively. The first detectible serology marker was Ab, followed by IgM and IgG, with a median seroconversion time of 15, 18 and 20 days post exposure (d.p.e) or 9, 10 and 12 days post onset (d.p.o), respectively. The antibody levels increased rapidly beginning at 6 d.p.o. and were accompanied by a decline in viral load. For patients in the early stage of illness (0-7 d.p.o), Ab showed the highest sensitivity (64.1%) compared to IgM and IgG (33.3% for both, p<0.001). The sensitivities of Ab, IgM and IgG increased to 100%, 96.7% and 93.3% 2 weeks later, respectively. When the same antibody type was detected, no significant difference was observed between enzyme-linked immunosorbent assays and other forms of immunoassays. CONCLUSIONS A typical acute antibody response is induced during SARS-CoV-2 infection. Serology testing provides an important complement to RNA testing in the later stages of illness for pathogenic specific diagnosis and helpful information to evaluate the adapted immunity status of patients.

Title: Serological studies of transmissible gastroenteritis in Great Britain, using a competitive ELISA. Content: A competitive ELISA which differentiates between transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) was used to detect non-neutralising antibodies to the peplomer protein of TGEV in porcine sera. The test was shown to be TGEV specific, having a relative specificity of 100 per cent, and to have a relative sensitivity of 94.9 per cent when compared with the virus neutralisation test. The prevalence of TGEV in Great Britain is low; only 0.6 per cent of sows sampled in 1990 were seropositive to TGEV. Seroconversion to the virus neutralisation test occurred in a closed herd in 1984, with no apparent spread, but later testing by the ELISA did not detect any blocking antibodies. The possibility of the existence of a less contagious strain of PRCV is discussed. All British isolates of TGEV tested by the indirect fluorescent antibody test were recognised by the monoclonal antibody 1D.B12, the indicator antibody in the ELISA.

Title: Diagnostic value and dynamic variance of serum antibody in coronavirus disease 2019 Content: OBJECTIVE: To investigate the diagnostic value of serological testing and dynamic variance of serum antibody in coronavirus disease 2019 (COVID-19). METHODS: This study retrospectively included 43 patients with a laboratory-confirmed infection and 33 patients with a suspected infection, in whom the disease was eventually excluded. The IgM/IgG titer of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was measured by chemiluminescence immunoassay analysis. RESULTS: Compared to molecular detection, the sensitivities of serum IgM and IgG antibodies to diagnose COVID-19 were 48.1% and 88.9%, and the specificities were 100% and 90.9%, respectively.In the COVID-19 group, the IgM-positive rate increased slightly at first and then decreased over time; in contrast, the IgG-positive rate increased to 100% and was higher than IgM at all times. The IgM-positive rate and titer were not significantly different before and after conversion to virus-negative. The IgG-positive rate was up to 90% and not significantly different before and after conversion to virus-negative. However, the median IgG titer after conversion to virus-negative was double that before, and the difference was significant. CONCLUSIONS: Viral serological testing is an effective means of diagnosis for SARS-CoV-2 infection. The positive rate and titer variance of IgG are higher than those of IgM in COVID-19.

Title: Serology characteristics of SARS-CoV-2 infection since exposure and post symptom onset Content: BACKGROUND: Timely diagnosis of SARS-CoV-2 infection is a prerequisite for treatment and prevention. The serology characteristics and complement diagnosis value of the antibody test to RNA test need to be demonstrated. METHOD: Serial sera of 80 patients with PCR-confirmed COVID-19 were collected at the First Affiliated Hospital of Zhejiang University, China. Total antibody (Ab), IgM and IgG antibodies against SARS-CoV-2 were detected, and the antibody dynamics during the infection were described. RESULTS: The seroconversion rates for Ab, IgM and IgG were 98.8%, 93.8% and 93.8%, respectively. The first detectible serology marker was Ab, followed by IgM and IgG, with a median seroconversion time of 15, 18 and 20â days post exposure (d.p.e) or 9, 10 and 12â days post onset (d.p.o), respectively. The antibody levels increased rapidly beginning at 6â d.p.o. and were accompanied by a decline in viral load. For patients in the early stage of illness (0-7â d.p.o), Ab showed the highest sensitivity (64.1%) compared to IgM and IgG (33.3% for both, p<0.001). The sensitivities of Ab, IgM and IgG increased to 100%, 96.7% and 93.3% 2â weeks later, respectively. When the same antibody type was detected, no significant difference was observed between enzyme-linked immunosorbent assays and other forms of immunoassays. CONCLUSIONS: A typical acute antibody response is induced during SARS-CoV-2 infection. Serology testing provides an important complement to RNA testing in the later stages of illness for pathogenic specific diagnosis and helpful information to evaluate the adapted immunity status of patients.

Title: Test, test, test for COVID-19 antibodies: the importance of sensitivity, specificity and predictive powers Content: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody tests of varying specificity and sensitivity are now available. For informing individuals whether they have had coronavirus disease 2019 (COVID-19), they need to be very accurate. For measuring population prevalence of past infection, the numbers of false positives and negatives need to be roughly equal. With a series of worked examples for a notional population of 100,000 people, we show that even test systems with a high specificity can yield a large number of false positive results, especially where the population prevalence is low. For example, at a true population prevalence of 5%, using a test with 99% sensitivity and specificity, 16% of positive results will be false and thus 950 people will be incorrectly informed they have had the infection. Further confirmatory testing may be needed. Giving false reassurance on which personal or societal decisions might be based could be harmful for individuals, undermine public confidence and foster further outbreaks.

Title: In vitro diagnostics of coronavirus disease 2019: Technologies and application Content: Laboratory-based diagnostic measures including virological and serological tests are essential for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Real-time reverse transcription-polymerase chain reactions (rRT-PCR) can detect SARS-COV-2 by targeting open reading frame-1 antibodies (ORF1ab), envelope protein, nucleocapsid protein, RNA-dependent RNA polymerase genes, and the N1, N2, and N3 (3N) target genes. Therefore, rRT-PCR remains the primary method of diagnosing SARS-CoV-2 despite being limited by false-negative results, long turnaround, complex protocols, and a need for skilled personnel. Serological diagnosis of coronavirus disease 2019 (COVID-19) is simple and does not require complex techniques and equipment, rendering it suitable for rapid detection and massive screening. However, serological tests cannot confirm SARS-CoV-2, and results will be false-negative when antibody concentrations fall below detection limits. Balancing the increased use of laboratory tests, risk of testing errors, need for tests, burden on healthcare systems, benefits of early diagnosis, and risk of unnecessary exposure is a significant and persistent challenge in diagnosing COVID-19.

Title: A serological assay to detect SARS-CoV-2 seroconversion in humans Content: Here, we describe a serological enzyme-linked immunosorbent assay for the screening and identification of human SARS-CoV-2 seroconverters. This assay does not require the handling of infectious virus, can be adjusted to detect different antibody types in serum and plasma and is amenable to scaling. Serological assays are of critical importance to help define previous exposure to SARS-CoV-2 in populations, identify highly reactive human donors for convalescent plasma therapy and investigate correlates of protection.

Title: A serological assay to detect SARS-CoV-2 seroconversion in humans. Content: Here, we describe a serological enzyme-linked immunosorbent assay for the screening and identification of human SARS-CoV-2 seroconverters. This assay does not require the handling of infectious virus, can be adjusted to detect different antibody types in serum and plasma and is amenable to scaling. Serological assays are of critical importance to help define previous exposure to SARS-CoV-2 in populations, identify highly reactive human donors for convalescent plasma therapy and investigate correlates of protection.

Title: Detection of bovine coronavirus by enzyme-linked immunosorbent assay using monoclonal antibodies. Content: A sensitive and highly specific enzyme-linked immunosorbent assay (ELISA) system for detection of bovine coronavirus was developed using monoclonal antibodies, which showed both neutralization and hemagglutination inhibition activities. A monoclonal antibody was coated onto microplates for capturing antigen and a mixture of two monoclonal antibodies served as detecting antibodies. Using this assay, 40 of 202 fecal specimens from 29 herds were shown to be positive for viral antigen. From 10 ELISA positive specimens from 5 herds, the virus was isolated after several passages on human rectal adenocarcinoma (HRT-18) cell culture.

Title: Enzyme-linked immunosorbent assay for the detection of canine coronavirus and its antibody in dogs Content: Abstract Two methods of enzyme-linked immunosorbent assay (ELISA) were developed for the diagnosis of canine coronavirus (CCV) infection in dogs. One ELISA, in which CCV-infected CRFK cell lysate is used as antigen, is for the detection and titration of antibody against CCV, and the other ELISA uses the double antibody sandwich method for the detection of CCV antigen. The first ELISA procedure demonstrated antibody responses in dogs inoculated with CCV, as did the virus neutralization test; the second ELISA detected specific CCV antigen in feces and organ homogenates of inoculated dogs.

Title: Antibody Detection and Dynamic Characteristics in Patients with COVID-19 Content: BACKGROUND: The corona virus disease 2019 (COVID-19) caused by the corona virus 2 (SARS-CoV-2) has been rapidly spreading nationwide and abroad. A serologic test to identify antibody dynamics and response to SARS-CoV-2 was developed. METHODS: The antibodies against SARS-CoV-2 were detected by an enzyme-linked immunosorbent assay (ELISA) based on the recombinant nucleocapsid protein of SARS-CoV-2 in patients with confirmed or suspected COVID-19 at 3-40 days after symptom onset. The gold standard for COVID-19 diagnosis was nucleic acid testing for SARS-CoV-2 by RT-PCR. The serodiagnostic power of the specific IgM and IgG antibodies against SARS-CoV-2 was investigated in terms of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and consistency rate. RESULTS: The seroconversion of specific IgM and IgG antibodies were observed as early as the 4(th) day after symptom onset. In the confirmed patients with COVID-19, sensitivity, specificity, PPV, NPV, and consistency rate of IgM were 77.3% (51/66), 100%, 100%, 80.0%, and 88.1%, and those of IgG were 83.3.3% (55/66), 95.0%, 94.8%, 83.8%, and 88.9 %. In patients with suspected COVID-19, sensitivity, specificity, PPV, NPV, and consistency rate of IgM were 87.5% (21/24), 100%, 100%, 95.2%, and 96.4%, and those of IgG were 70.8% (17/24), 96.6%, 85.0%, 89.1%, and 88.1%. Both antibodies performed well in serodiagnosis for COVID-19 rely on great specificity. CONCLUSIONS: The antibodies against SARS-CoV-2 can be detected in the middle and later stage of the illness. Antibody detection may play an important role in the diagnosis of COVID-19 as complement approach for viral nucleid acid assays.

Title: Development of a competitive enzyme-linked immunosorbent assay for detection of turkey coronavirus antibodies. Content: A competitive enzyme-linked immunosorbent assay (cELISA) was developed for detection of turkey coronavirus (TCV) antibodies. The cELISA utilized a recombinant baculovirus (Autographa californica nuclear polyhedrosis virus)-expressed TCV nucleocapsid (N) protein and biotin-labeled TCV N protein-specific monoclonal antibody. Sensitivity and specificity of the cELISA for detection of TCV antibodies were determined by comparison with the indirect fluorescent antibody test (IFAT) with 1269 reference, experimentally derived, and field-origin sera. Sera with discordant cELISA and IFAT results were further evaluated by western immunoblot analyses. The cELISA detected antibodies specific for TCV and infectious bronchitis virus, a closely related coronavirus, but did not detect antibodies specific for other avian viruses. A high degree of concordance was observed between the cELISA and IFAT; sensitivity and specificity of the cELISA relative to IFAT were 92.9% and 96.2%, respectively. Western immunoblot analyses provided additional evidence of cELISA specificity. The findings indicate that the cELISA is a rapid, sensitive, and specific serologic test for detection of TCV antibodies in turkeys.