are there serological tests that detect antibodies to coronavirus?

Severe Acute Respiratory Syndrome Coronavirus 2-Specific Antibody Responses in Coronavirus Disease Patients

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.

Severe Acute Respiratory Syndrome Coronavirus 2−Specific Antibody Responses in Coronavirus Disease Patients

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.

Serological differentiation between COVID-19 and SARS infections.

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.

Serological differentiation between COVID-19 and SARS infections

In response to the coronavirus disease 2019 (COVID-19) outbreak, caused by SARS-CoV-2, multiple diagnostic tests are required for acute disease diagnosis, contact tracing, monitoring 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. Unlike PCR tests which are highly specific, cross-reactivity is a major challenge for COVID-19 antibody tests considering there are six other coronaviruses known to infect humans. SARS-CoV is genetically related to SARS-CoV-2 sharing approximately 80% sequence identity and both belong to the species SARS related coronavirus in the genus Betacoronavirus of family Coronaviridae. We developed and compared the performance of four different serological tests to comprehensively assess the cross-reactivity between COVID-19 and SARS patient sera. There is significant cross-reactivity when N protein of either virus is used. The S1 or RBD regions from the spike (S) protein offers better specificity. Amongst the different platforms, capture ELISA performed best. We found that SARS survivors all have significant levels of antibodies remaining in their blood 17 years after infection. Anti-N antibodies waned more than anti-RBD antibodies, and the latter is known to play a more important role in providing protective immunity.

Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray

The current practice for diagnosis of SARS-CoV-2 infection relies on PCR testing of nasopharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk. This testing strategy likely underestimates the true prevalence of infection, creating the need for serologic methods to detect infections missed by the limited testing to date. Here, we describe the development 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 preliminary study of human sera collected prior to the SARS-CoV-2 pandemic demonstrates overall high IgG reactivity to common human coronaviruses and low IgG reactivity to epidemic coronaviruses including SARS-CoV-2, with some cross-reactivity of conserved antigenic domains including S2 domain of spike protein and nucleocapsid protein. This array can be used to answer outstanding questions regarding SARS-CoV-2 infection, including whether baseline serology for other coronaviruses impacts disease course, how the antibody response to infection develops over time, and what antigens would be optimal for vaccine development.

Serological assays for emerging coronaviruses: Challenges and pitfalls

Abstract More than a decade after the emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002/2003 the occurrence of a novel CoV termed Middle East respiratory syndrome (MERS) CoV challenges researchers and public health authorities. To control spread and finally contain novel viruses, rapid identification and subsequent isolation of infected individuals and their contacts is of utmost importance. Next to methods for nucleic acid detection, validated serological assays are particularly important as the timeframe for antibody detection is less restricted. During the SARS-CoV epidemic a wide variety of serological diagnostic assays were established using multiple methods as well as different viral antigens. Even though the majority of the developed assays showed high sensitivity and specificity, numerous studies reported on cross-reactive antibodies to antigens from wide-spread common cold associated CoVs. In order to improve preparedness and responsiveness during future outbreaks of novel CoVs, information and problems regarding serological diagnosis that occurred during the SARS-CoV should be acknowledged. In this review we summarize the performance of different serological assays as well as the applicability of the two main applied antigens (spike and nucleocapsid protein) used during the SARS-CoV outbreak. We highlight challenges and potential pitfalls that occur when dealing with a novel emerging coronavirus like MERS-CoV. In addition we describe problems that might occur when animal sera are tested in serological assays for the identification of putative reservoirs. Finally, we give a recommendation for a serological testing scheme and outline necessary improvements that should be implemented for a better preparedness.

Immunological assays for SARS-CoV-2: an analysis of available commercial tests to measure antigen and antibodies

The rapid spread of SARS-CoV-2 coronavirus infection has led to the development of molecular and serologic tests in a short period of time. While tests such as RT-PCR have applications in the immediate diagnosis revealing the presence of the virus, serological tests can be used to determine previous exposure to the virus and complement acute diagnosis. Antibody production can occur as early as 5 days post-infection. Both IgM and IgG specific anti-SARS-COV-2 antibodies can be a useful tool to test faster and larger groups of individuals. The objective of this study was to carry out a review of the different serological tests offered to detect antigen or antibodies against SARS-CoV-2. This information should be useful for decision takers in different countries to choose a test according to their needs. Based on web pages that listed serological assays, we found 226 coming from 20 countries, the majority are indirect tests for specific antibodies detection (n 180) and use immunochromatography methods (n 110) with samples coming from blood-derived products (n 105). Measuring IgM/IgG at the same time (n 112) and a procedure time of <20 min (n 83) are the most common. The overall average sensitivity was 91.8% and specificity was 97%. Most of the tests are currently for in vitro diagnosis (IVD). This information gathered could change day by day due to the expedite process of production and emergency of authorization use.

Serological responses in patients with severe acute respiratory syndrome coronavirus infection and cross-reactivity with human coronaviruses 229E, OC43, and NL63.

The serological response profile of severe acute respiratory syndrome (SARS) coronavirus (CoV) infection was defined by neutralization tests and subclass-specific immunofluorescent (IF) tests using serial sera from 20 patients. SARS CoV total immunoglobulin (Ig) (IgG, IgA, and IgM [IgGAM]) was the first antibody to be detectable. There was no difference in time to seroconversion between the patients who survived (n = 14) and those who died (n = 6). Although SARS CoV IgM was still detectable by IF tests with 8 of 11 patients at 7 months postinfection, the geometric mean titers dropped from 282 at 1 month postinfection to 19 at 7 months (P = 0.001). In contrast, neutralizing antibody and SARS CoV IgGAM and IgG antibody titers remained stable over this period. The SARS CoV antibody response was sometimes associated with an increase in preexisting IF IgG antibody titers for human coronaviruses OC43, 229E, and NL63. There was no change in IF IgG titer for virus capsid antigen from the herpesvirus that was used as an unrelated control, Epstein-Barr virus. In contrast, patients who had OC43 infections, and probably also 229E infections, without prior exposure to SARS CoV had increases of antibodies specific for the infecting virus but not for SARS CoV. There is a need for awareness of cross-reactive antibody responses between coronaviruses when interpreting IF serology.

Coronavirus infection of spotted hyenas in the Serengeti ecosystem

Abstract Sera from 38 free-ranging spotted hyenas (Crocuta crocuta) in the Serengeti ecosystem, Tanzania, were screened for exposure to coronavirus of antigenic group 1. An immunofluorescence assay indicated high levels of exposure to coronavirus among Serengeti hyenas: 95% when considering sera with titer levels of ≥1:10 and 74% when considering sera with titer levels of ≥1:40. Cubs had generally lower mean titer levels than adults. Exposure among Serengeti hyenas to coronavirus was also confirmed by a serum neutralisation assay and an ELISA. Application of RT-PCR to 27 fecal samples revealed viral RNA in three samples (11%). All three positive fecal samples were from the 15 juvenile animals (<24 months of age) sampled, and none from the 12 adults sampled. No viral RNA was detected in tissue samples (lymph node, intestine, lung) from 11 individuals. Sequencing of two amplified products from the S protein gene of a positive sample revealed the presence of coronavirus specific RNA with a sequence homology to canine coronavirus of 76 and 78% and to feline coronavirus type II of 80 and 84%, respectively. Estimation of the phylogenetic relationship among coronavirus isolates indicated considerable divergence of the hyena variant from those in European, American and Japanese domestic cats and dogs. From long-term observations of several hundred known individuals, the only clinical sign in hyenas consistent with those described for coronavirus infections in dogs and cats was diarrhea. There was no evidence that coronavirus infection in hyenas caused clinical signs similar to feline infectious peritonitis in domestic cats or was a direct cause of mortality in hyenas. To our knowledge, this is the first report of coronavirus infection in Hyaenidae.

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.

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.

Antigenic relationships amongst coronaviruses

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.

SARS-CoV-2 specific antibody responses in COVID-19 patients

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.

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

Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome.

To identify a major antigenic determinant for use in the development of a rapid serological diagnostic test for severe acute respiratory syndrome (SARS) coronavirus infection and to study the immune response during SARS coronavirus infection in humans, we cloned the full length and six truncated fragments of the nucleocapsid gene, expressed them, and purified them as glutathione S-transferase-tagged recombinant proteins. The reactivities of the recombinant proteins to a panel of antibodies containing 33 SARS coronavirus-positive sera and 66 negative sera and to antibodies against other animal coronaviruses were screened. A truncated 195-amino-acid fragment from the C terminus of the nucleocapsid protein (N195) was identified that had a strong ability to detect antibodies against SARS coronavirus. No cross-reaction was found between the N195 protein and antibodies against chicken, pig, and canine coronaviruses. The N195 protein was used to develop a Western blot assay to detect antibodies against SARS coronavirus in 274 clinically blinded samples. The specificity and sensitivity of this test were 98.3 and 90.9%, respectively. The correlation between our Western blotting assay and an immunofluorescence assay (IFA) was also analyzed. The results of our Western blot assay and IFA for the detection of SARS coronavirus-positive sera were the same. Thus, the N195 protein was identified as a suitable protein to be used as an antigen in Western blot and other possible assays for the detection of SARS coronavirus infection.

Dynamic profile for the detection of anti-SARS-CoV-2 antibodies using four immunochromatographic assays

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.

Serologic and Molecular Biologic Methods for SARS-associated Coronavirus Infection, Taiwan

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.

Serologic evidence of coronavirus infection in New York and New England dairy cattle with winter dysentery.

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.

Detection of feline coronavirus infection in captive cheetahs (Acinonyx jubatus) by polymerase chain reaction.

Feline coronavirus genetic elements were detected by polymerase chain reaction from blood, fecal samples, and effusive fluid collected from 33 cheetahs in the U.S.A. Feline coronavirus-specific serum antibodies were also measured by indirect immunofluorescence. Ten cheetahs were positive for viral shedding by polymerase chain reaction, whereas 13 were seropositive by immunofluorescence. Results of serology did not consistently correlate with shedding of virus, and the capture antigen used for detection of feline coronavirus-specific antibodies had a significant impact on results. Testing of samples from one population over a 1-yr period indicated chronic infection in some animals. These relatively healthy carrier animals were a source of virus for contact animals. Screening programs in cheetah populations for feline coronavirus infection may be most reliable if a combination of serologic analysis and viral detection by polymerase chain reaction is used.

Abstract 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 antiboy 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.

[Seroepidemiological study of coronavirus infection in children and adults in St. Petersburg].

As the result of prolonged (17 years) observations of patients with acute respiratory infections hospitalized in basic departments of clinics of the Research Institute of Influenza, coronavirus infection was found to be the cause of respiratory diseases, on the average, in 12% of cases (in some years in 6.8% to 28.6% of cases). The analysis of extensive morbidity rates among different age groups of the population showed that children were affected by coronavirus infection 5-7 times more often than adults. Three year cycles of this infection were established. The periods of coronaviruses activation were accompanied by their detection in patient material by electron-microscopy, a sharp increase of immune response of patients as well as in the number of nosocomial infections and the proportion of the monoinfection of the coronavirus nature. Coronaviruses played the leading role among other viruses in the etiology of hospital respiratory infections. Mucosal antibodies to coronaviruses in the secretions of the nasal cavity proved to be more important than serum antibodies not only in protection from infection, but also in the pattern of clinical manifestations of the disease.

SARS-CoV-2 assays to detect functional antibody responses that block ACE2 recognition in vaccinated animals and infected patients

SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) has caused a global pandemic of COVID-19 resulting in cases of mild to severe respiratory distress and significant mortality. The global outbreak of this novel coronavirus has now infected >8 million people worldwide with >2 million cases in the US (June 17th, 2020). There is an urgent need for vaccines and therapeutics to combat the spread of this coronavirus. Similarly, the development of diagnostic and research tools to determine infection and vaccine efficacy are critically needed. Molecular assays have been developed to determine viral genetic material present in patients. Serological assays have been developed to determine humoral responses to the spike protein or receptor binding domain (RBD). Detection of functional antibodies can be accomplished through neutralization of live SARS-CoV2 virus, but requires significant expertise, an infectible stable cell line, a specialized BioSafety Level 3 (BSL-3) facility. As large numbers of people return from quarantine, it is critical to have rapid diagnostics that can be widely adopted and employed to assess functional antibody levels in the returning workforce. This type of surrogate neutralization diagnostic can also be used to assess humoral immune responses induced in patients from the large number of vaccine and immunotherapy trials currently on-going. Here we describe a rapid serological diagnostic assay for determining antibody receptor blocking and demonstrate the broad utility of the assay by measuring the antibody functionality of sera from small animals and non-human primates immunized with an experimental SARS-CoV-2 vaccine and using sera from infected patients.

Simultaneous detection of five major serotypes of Avian coronavirus by a multiplex microsphere-based assay.

Avian coronavirus (commonly known as Infectious bronchitis virus [IBV]) is of major economic importance to commercial chicken producers worldwide. Due to the existence of multiple serotypes and variants of the virus that do not cross-protect, it is important to diagnose circulating serotypes and choose the right vaccine type for successful protection. In an effort to improve conventional diagnostic tests, a microsphere-based assay was developed and evaluated for simultaneous detection of the most common IBV vaccine serotypes in the United States: Arkansas (Ark), Connecticut (Conn), Massachusetts (Mass), Delaware (DE072), and Georgia 98 (GA98). The analytical specificity and sensitivity, and diagnostic specificity and sensitivity, were evaluated. The microsphere-based assay was highly specific to designated serotypes and generated reproducible data. Comparing the microsphere-based assay to nucleotide sequencing, the 2 methods agreed more than 93% (kappa value > .77). In addition, the microsphere-based assay could detect coinfections in clinical samples. The results demonstrate the utility of the microsphere-based assay as a rapid and accurate diagnostic tool with the potential for high throughput diagnosis.

Efficacy of Serology Testing in Predicting Reinfection in Patients with SARS-CoV-2

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.

Testing COVID-19 tests faces methodological challenges

In battling the COVID-19 pandemic, testing is essential. The detection of viral RNA allows the identification of infected persons, while the detection of antibodies may reveal a response to a previous infection. Tests for coronavirus should be rigorously evaluated in terms of their analytical and clinical performance. This poses not only logistic challenges, but also methodological ones. Some of these are generic for the diagnostic accuracy paradigm, while others are more specific for tests for viruses. Problematic for evaluations of the clinical performance of tests for viral RNA is the absence of an independent reference standard. Many studies lack rigor in terms of the recruitment of study participants. Study reports are often insufficiently informative, which makes it difficult to assess the applicability of study findings. Attempts to summarize the performance of these tests in terms of a single estimate of the clinical sensitivity fails to do justice to the identifiable sources of the large heterogeneity in mechanisms for generating false negative results.

Serological relation between calf diarrhea coronavirus and hemagglutinating encephalomyelitis virus

Neutralizing (NT) and hemagglutination-inhibiting (HI) antibodies to calf diarrhea coronavirus (CDCV) and hemagglutinating encephalomyelitis virus of swine (HEV) (strain 67 N) were detected in high proportions of normal adult cattle and pigs in Japan. Since comparison of NT and HI titers in the serum samples suggested an antigenic difference between the viruses, cross NT and HI tests of these viruses were carried out with antisera raised in rabbits. The homologous NT titers were markedly higher than the heterologous titers. In HI tests essentially the same results were obtained. These findings indicate the presence of a marked difference in antigenic make-up between CDCV and HEV. NT and HI tests can clearly differentiate the viruses, although there is some cross reaction.

Diagnostic value and dynamic variance of serum antibody in coronavirus disease 2019

Abstract 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.

Efficacy of Serology Testing in Predicting Reinfection in Patients with SARS-CoV-2.

Abstract In battling the COVID-19 pandemic, testing is essential. The detection of viral RNA allows the identification of infected persons, while the detection of antibodies may reveal a response to a previous infection. Tests for coronavirus should be rigorously evaluated in terms of their analytical and clinical performance. This poses not only logistic challenges, but also methodological ones. Some of these are generic for the diagnostic accuracy paradigm, while others are more specific for tests for viruses. Problematic for evaluations of the clinical performance of tests for viral RNA is the absence of an independent reference standard. Many studies lack rigor in terms of the recruitment of study participants. Study reports are often insufficiently informative, which makes it difficult to assess the applicability of study findings. Attempts to summarize the performance of these tests in terms of a single estimate of the clinical sensitivity fails to do justice to the identifiable sources of the large heterogeneity in mechanisms for generating false negative results.

Diagnostic accuracy of an automated chemiluminescent immunoassay for anti-SARS-CoV-2 IgM and IgG antibodies: an Italian experience

A pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spreading throughout the world. Though molecular diagnostic tests are the gold standard for COVID-19, serological testing is emerging as a potential surveillance tool, in addition to its complementary role in COVID-19 diagnostics. Indubitably quantitative serological testing provides greater advantages than qualitative tests but today there is still little known about serological diagnostics and what the most appropriate role quantitative tests might play. Sixty-one COVID-19 patients and 64 patients from a control group were tested by iFlash1800 CLIA analyzer for anti-SARS CoV-2 antibodies IgM and IgG. All COVID-19 patients were hospitalized in San Giovanni di Dio Hospital (Florence, Italy) and had a positive oro/nasopharyngeal swab reverse-transcription polymerase chain reaction result. The highest sensitivity with a very good specificity performance was reached at a cutoff value of 10.0 AU/mL for IgM and of 7.1 for IgG antibodies, hence near to the manufacturer's cutoff values of 10 AU/mL for both isotypes. The receiver operating characteristic curves showed area under the curve values of 0.918 and 0.980 for anti-SARS CoV-2 antibodies IgM and IgG, respectively. iFlash1800 CLIA analyzer has shown highly accurate results for the anti-SARS-CoV-2 antibodies profile and can be considered an excellent tool for COVID-19 diagnostics.

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.

[Acute respiratory disease caused by coronaviruses].

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%).

Detection by radioimmunoassay and enzyme-linked immunosorbent assay of coronavirus antibodies in bovine serum and lacteal secretions.

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.

Serodiagnostics for Severe Acute Respiratory Syndrome–Related Coronavirus-2: A Narrative Review

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.

Serological Screening for Coronavirus Infections in Cats

Coronaviruses (CoVs) are widespread among mammals and birds and known for their potential for cross-species transmission. In cats, infections with feline coronaviruses (FCoVs) are common. Several non-feline coronaviruses have been reported to infect feline cells as well as cats after experimental infection, supported by their ability to engage the feline receptor ortholog for cell entry. However, whether cats might become naturally infected with CoVs of other species is unknown. We analyzed coronavirus infections in cats by serological monitoring. In total 137 cat serum samples and 25 FCoV type 1 or type 2-specific antisera were screened for the presence of antibodies against the S1 receptor binding subunit of the CoV spike protein, which is immunogenic and possesses low amino acid sequence identity among coronavirus species. Seventy-eight sera were positive for antibodies that recognized one or more coronavirus S1s whereas 1 serum exclusively reacted with human coronavirus 229E (HCoV-229E) and two sera exclusively reacted with porcine delta coronavirus (PDCoV). We observed antigenic cross-reactivity between S1s of type 1 and type 2 FCoVs, and between FCoV type 1 and porcine epidemic diarrhea virus (PEDV). Domain mapping of antibody epitopes indicated the presence of conserved epitope(s) particularly in the CD domains of S1. The cross-reactivity of FCoV type 1 and PEDV was also observed at the level of virus neutralization. To conclude, we provide the first evidence of antigenic cross-reactivity among S1 proteins of coronaviruses, which should be considered in the development of serological diagnoses. In addition, the potential role of cats in cross-species transmission of coronaviruses cannot be excluded.

Serodiagnostics for Severe Acute Respiratory Syndrome-Related Coronavirus-2: A Narrative Review

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.

Coronavirus vaccine developers wary of errant antibodies.

A serological assay to detect SARS-CoV-2 seroconversion in humans

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.

B-cell responses in patients who have recovered from severe acute respiratory syndrome target a dominant site in the S2 domain of the surface spike glycoprotein.

Severe acute respiratory syndrome (SARS) is a recently emerged infectious disease caused by a novel strain of coronavirus. Examination of the immune responses of patients who have recovered from SARS should provide important information for design of a safe and effective vaccine. We determined the continuous viral epitopes targeted by antibodies in plasma samples from convalescent SARS patients through biopanning with a vast M13 phage display dodecapeptide library. These epitopes converged to very short peptide fragments, one on each of the structural proteins spike and nucleocapsid and the nonstructural proteins 3a, 9b, and nsp 3. Immunoassays found that most of the patients who had recovered from SARS developed complementary antibodies to the epitope-rich region on the spike S2 protein, indicating that this is an immunodominant site on the viral envelope comprising the spike, matrix, and small envelope glycoproteins. These S2-targeting antibodies were shown to effectively neutralize the coronavirus, indicating that they provided protective immunity to help the patients recover from the viral infection. These results suggest that the SARS coronavirus might have an antigenic profile distinct from those of other human or animal coronaviruses. Due to the tested safety and protective effects of the convalescent-phase serological antibodies, identification of their complementary antigens may enable the design of an epitope-based vaccine to prevent potential antibody-mediated immunopathology.

Antibody Detection and Dynamic Characteristics in Patients with COVID-19

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.

Comparison of serologic techniques for the detection of antibodies against feline coronaviruses.

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.

Monoclonal antibody capture enzyme-linked immunosorbent assay for detection of bovine enteric coronavirus.

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.

[Rapid point-of-care serology testing for sars-cov-2].

Increasing evidence indicates immunity against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) after covid-19, but it remains unclear for how long the protection remains. Serology testing seems to have a higher sensitivity than molecular diagnostics from 8 days after onset of symtoms, and should be part of risk assessment and epidemiological studies of COVID-19. The performance of commercial serological point-of-care (POC) lateral flow tests are highly manufacturer-dependant. Low sensitivity increases the risk of false negative results and could result in unnecessary quarantine of test persons with developed antibodies. Low specificity increases the risk of false positive results and could lead to false assumptions of immunity. Carefully selected serological POC tests for sars-cov-2 can be used in large scale testing but should only be used by licensed medical staff able to understand their limitations and interpret the results.

Nasopharyngeal and Oropharyngeal Swabs, And/Or Serology for SARS COVID-19: What Are We Looking For?

Governments and clinicians that were fully involved in the dramatic SARS-CoV-2 outbreak during the last few weeks in Italy (and more or less all over the world) are fiercely debating the use of methods for screening this viral infection. Thus, all countries are employing a lot of resources in order to test more and more subjects. For this purpose, there are different strategies, based on either direct or indirect tests. Among the first category, the main assays used for SARS-CoV-2 are based on a real-time reverse transcriptase polymerase chain reaction (RT-PCR). Such tests can be performed on nasopharyngeal and oropharyngeal swabs for the categories of those with symptoms and those potentially exposed. In order to integrate the molecular assays in the diagnosis of SARS-CoV-2, a wide range of serology immunoassays (IAs) have also been developed. If we want to identify "immune" people in order to let them to come back to work, serology is the best (and probably the only) approach.

Severe acute respiratory syndrome coronavirus 2, original antigenic sin, and antibody-dependent enhancement: ménage à trois.

BACKGROUND Shortly after its emergence in December 2019, the coronavirus disease 2019 (COVID-19) was declared as a pandemic by the World Health Organization. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the seventh member of the Coronaviridae family of viruses that causes disease in humans. THE PROBLEM Despite the established role of molecular diagnostics, COVID-19 serodiagnosis remains a poorly discovered and enigmatic area. Although there are numerous commercial serological products available globally, there is a severe paucity of high-quality peer-reviewed literature on their true performance characteristics. That being said, publications including in-house developed serological tests started to shed light on the kinetics of the humoral response. SUMMARY In spite of intense focus of assessing the performance characteristics of the commercially-available kits, the main issue remains rather invisible, that is, lack of solid science behind COVID-19 serology its clinical usefulness thereof. This short review summarizes the key points as to why COVID-19 is not jest ready to fly. PURPOSE OF REVIEW Despite having been mentioned as a testing option, COVID-19 serology has significant shortcomings that needs discussing. This short review is meant to shed light on one of those aspects.

Characterization of novel monoclonal antibodies against the MERS-coronavirus spike protein and their application in species-independent antibody detection by competitive ELISA

Abstract Since discovering the Middle East respiratory syndrome coronavirus (MERS-CoV) as a causative agent of severe respiratory illness in the Middle East in 2012, serological testing has been conducted to assess antibody responses in patients and to investigate the zoonotic reservoir of the virus. Although the virus neutralization test is the gold standard assay for MERS diagnosis and for investigating the zoonotic reservoir, it uses live virus and so must be performed in high containment laboratories. Competitive ELISA (cELISA), in which a labeled monoclonal antibody (MAb) competes with test serum antibodies for target epitopes, may be a suitable alternative because it detects antibodies in a species-independent manner. In this study, novel MAbs against the spike protein of MERS-CoV were produced and characterized. One of these MAbs was used to develop a cELISA. The cELISA detected MERS-CoV-specific antibodies in sera from MERS-CoV-infected rats and rabbits immunized with the spike protein of MERS-CoV. The MAb-based cELISA was validated using sera from Ethiopian dromedary camels. Relative to the neutralization test, the cELISA detected MERS-CoV-specific antibodies in 66 Ethiopian dromedary camels with a sensitivity and specificity of 98% and 100%, respectively. The cELISA and neutralization test results correlated well (Pearson’s correlation coefficients=0.71–0.76, depending on the cELISA serum dilution). This cELISA may be useful for MERS epidemiological investigations on MERS-CoV infection.

Diagnostic methods and potential portable biosensors for coronavirus disease 2019

Timely detection and diagnosis are urgently needed to guide epidemiological measures, infection control, antiviral treatment, and vaccine research. In this review, biomarkers/indicators for diagnosis of coronavirus disease 2019 (COVID-19) or detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the environment are summarized and discussed. It is concluded that the detection methods targeting antibodies are not suitable for screening of early and asymptomatic cases since most patients had an antibody response at about 10 days after onset of symptoms. However, antibody detection methods can be combined with quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) to significantly improve the sensitivity and specificity of diagnosis, and boost vaccine research. Fast, sensitive and accurate detection methods targeting antigens need to be developed urgently. Various specimens for diagnosis or detection are compared and analyzed. Among them, deep throat saliva and induced sputum are desired for RT-qPCR test or other early detection technologies. Chest computerized tomography (CT) scan, RT-qPCR, lateral flow immunochromatographic strip (LFICS) for diagnosis of COVID-19 are summarized and compared. Specially, potential electrochemical biosensor, surface enhanced Raman scattering (SERS)-based biosensor, and artificial intelligence (AI) assisted diagnosis of COVID-19 are emphasized. Finally, some commercialized portable detection device, current challenges and future directions are discussed.

Studies on the relationship between coronaviruses from the intestinal and respiratory tracts of calves

An immunofluorescence test on smears of nasal epithelial cells was used to detect coronavirus infection in the respiratory tract of calves. Thirteen gnotobiotic calves were infected with coronavirus isolates derived from faeces or respiratory material: virus was detected in faeces and nasal swabs from all animals. In 115 calves from a field survey, there was a significant association between coronavirus excretion from both respiratory and enteric routes in calves with diarrhoea. In a further 12 calves, at necropsy, the predilection sites for coronavirus growth were the distal small intestine, large intestine and the epithelia of the nasal cavity and trachea. Antigen was not found in lung tissue by immunofluoresence or immunoperoxidase staining. Infection with enteric coronavirus induced immunity to reinfection and to heterologous challenge with two coronavirus isolates derived from the respiratory tract. Nine coronaviruses were cultivated, cloned and antisera to three were prepared in pigs. There was complete virus neutralisation in tests with homologous sera and significant cross reactions with the eight other isolates which were of intestinal and respiratory origin. Thus, these bovine coronavirus isolates belonged to the same serotype despite the source of virus.

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 4th 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.

Longitudinal Monitoring of SARS-CoV-2 IgM and IgG Seropositivity to Detect COVID-19

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a novel beta-coronavirus that has recently emerged as the cause of the 2019 coronavirus pandemic (COVID-19). Polymerase chain reaction (PCR) based tests are optimal and recommended for the diagnosis of an acute SARS-CoV-2 infection. Serology tests for viral antibodies provide an important tool to diagnose previous exposure to the virus. Here we evaluate the analytical performance parameters of the Diazyme SARS-CoV-2 IgM/IgG serology assays and describe the kinetics of IgM and IgG seroconversion observed in patients with PCR confirmed COVID-19 who were admitted to our hospital. METHODS: We validated the performance of the Diazyme assay in 235 subjects to determine specificity. Subsequently, we evaluated the SARS-CoV-2 IgM and IgG seroconversion of 54 PCR confirmed COVID-19 patients and determined sensitivity of the assay at three different timeframes. RESULT: Sensitivity and specificity for detecting seropositivity at ≥ 15 days following a positive SARS-CoV-2 PCR result, was 100.0% and 98.7% when assaying for the panel of IgM and IgG. The median time to seropositivity observed for a reactive IgM and IgG result from the date of a positive PCR was 5 days (IQR: 2.75-9 days) and 4 days (IQR: 2.75-6.75 days), respectively. CONCLUSIONS: Our data demonstrates that the Diazyme IgM/IgG assays are suited for the purpose of detecting SARS-CoV-2 IgG and IgM in patients with suspected SARS-CoV-2 infections. For the first time, we report longitudinal data showing the evolution of seroconversion for both IgG and IgM in a cohort of acutely ill patients in the United States. We also demonstrate a low false positive rate in patients who were presumed to be disease free.

Coronavirus-associated antibodies in nasopharyngeal carcinoma and infectious mononucleosis

Coronavirus-like particles are found within the cytoplasm of NPC tumor cells, within the cytoplasm of the tumor cells of the regional metastases, and within tumor cells grown on nude mice. For the immunologic identification of the coronaviruses, the cultures of human tracheal epithelium (MRC-C) were used and inoculated with a known coronavirus strain. Whereas blood sera from NPC patients (n=73) contain significantly elevated antibody titers against corona viruses, unselected sera from patients without NPC showed a low antibody titer (n=83). Only patients suffering from infectious mononucleosis (n=40) showed a titer pattern similar to that of NPC patients. For demonstration of antigen-antibody reaction within the NPC tumor cell cytoplasm, sera with a high antibody content against coronaviruses deriving from other than NPC patients or anticoronavirus sera from rabbits were used. By indirect immunofluorescence, the NPC tumor cells showed a bright cytoplasmic fluorescence. No fluorescence was seen when tumor cells were exposed to human sera with known low or absent corona antibody titer or to normal rabbit sera. The results indicate that next to a DNA virus infection (EBV), an RNA virus infection (coronavirus) may play a role in NPC as well as in infectious mononucleosis.

Immunological detection of severe acute respiratory syndrome coronavirus by monoclonal antibodies.

In order to establish immunological detection methods for severe acute respiratory syndrome coronavirus (SARS-CoV), we established monoclonal antibodies directed against structural components of the virus. B cell hybridomas were generated from mice that were hyper-immunized with inactivated SARS-CoV virion. By screening 2,880 generated hybridomas, we established three hybridoma clones that secreted antibodies specific for nucleocapsid protein (N) and 27 clones that secreted antibodies specific for spike protein (S). Among these, four S-protein specific antibodies had in vitro neutralization activity against SARS-CoV infection. These monoclonal antibodies enabled the immunological detection of SARS-CoV by immunofluorescence staining, Western blot or immunohistology. Furthermore, a combination of monoclonal antibodies with different specificities allowed the establishment of a highly sensitive antigen-capture sandwich ELISA system. These monoclonal antibodies would be a useful tool for rapid and specific diagnosis of SARS and also for possible antibody-based treatment of the disease.

A comparison of the sensitivity and specificity of sialodacryoadenitis virus, Parker's rat coronavirus, and mouse hepatitis virus-infected cells as a source of antigen for the detection of antibody to rat coronaviruses

Sialodacryoadenitis virus (SDAV) and Parker's rat coronavirus (PRC) are two recognized viral strains which cause spontaneous disease in the laboratory rat. Currently there is no recognized practical procedure which will accurately differentiate infections with these strains. Using SDAV- and PRC-infected L-2 cells as the source of antigen, and sera from rats collected post inoculation with either of these viral strains, the indirect fluorescent antibody (IFA) procedure was used to determine whether antibody titers could be used to differentiate infections from the homologous and heterologous virus. There was no detectable difference in the sensitivity or specificity of these systems in detecting antibody to the homologous or heterologous virus. Thus there was no evidence that SDAV- and PRC-infected cells would serve to differentiate antibody to the homologous virus using the IFA technique. In addition, antibody titers were similar when mouse hepatitis virus (MHV)-infected cells were used as the source of antigen for the IFA technique. However, using MHV or SDAV-infected cells as the source of antigen, there was a significant difference in antibody titers to the homologous virus detected using the immunoenzyme technique.

[Serological diagnostic studies of the occurrence of coronavirus infections in cattle with respiratory diseases].

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.

Sensitivity in Detection of Antibodies to Nucleocapsid and Spike Proteins of Severe Acute Respiratory Syndrome Coronavirus 2 in Patients With Coronavirus Disease 2019

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), is associated with respiratory-related disease and death. Assays to detect virus-specific antibodies are important to understand the prevalence of infection and the course of the immune response. METHODS: Quantitative measurements of plasma or serum antibodies to the nucleocapsid and spike proteins were analyzed using luciferase immunoprecipitation system assays in 100 cross-sectional or longitudinal samples from patients with SARS-CoV-2 infection. A subset of samples was tested both with and without heat inactivation. RESULTS: At >14 days after symptom onset, antibodies against SARS-CoV-2 nucleocapsid protein showed 100% sensitivity and 100% specificity, whereas antibodies to spike protein were detected with 91% sensitivity and 100% specificity. Neither antibody levels nor the rate of seropositivity were significantly reduced by heat inactivation of samples. Analysis of daily samples from 6 patients with COVID-19 showed anti-nucleocapsid and spike protein antibodies appearing between days 8 and 14 after initial symptoms. Immunocompromised patients generally had a delayed antibody response to SARS-CoV-2, compared with immunocompetent patients. CONCLUSIONS: Antibody to the nucleocapsid protein of SARS-CoV-2 is more sensitive than spike protein antibody for detecting early infection. Analyzing heat-inactivated samples with a luciferase immunoprecipitation system assay is a safe and sensitive method for detecting SARS-CoV-2 antibodies.

Seroepidemiologic Study Designs for Determining SARS-COV-2 Transmission and Immunity.

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.

The receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that first emerged in late 2019 is responsible for a pandemic of severe respiratory illness. People infected with this highly contagious virus can present with clinically inapparent, mild, or severe disease. Currently, the virus infection in individuals and at the population level is being monitored by PCR testing of symptomatic patients for the presence of viral RNA. There is an urgent need for SARS-CoV-2 serologic tests to identify all infected individuals, irrespective of clinical symptoms, to conduct surveillance and implement strategies to contain spread. As the receptor binding domain (RBD) of the spike protein is poorly conserved between SARS-CoVs and other pathogenic human coronaviruses, the RBD represents a promising antigen for detecting CoV-specific antibodies in people. Here we use a large panel of human sera (63 SARS-CoV-2 patients and 71 control subjects) and hyperimmune sera from animals exposed to zoonotic CoVs to evaluate RBD's performance as an antigen for reliable detection of SARS-CoV-2-specific antibodies. By day 9 after the onset of symptoms, the recombinant SARS-CoV-2 RBD antigen was highly sensitive (98%) and specific (100%) for antibodies induced by SARS-CoVs. We observed a strong correlation between levels of RBD binding antibodies and SARS-CoV-2 neutralizing antibodies in patients. Our results, which reveal the early kinetics of SARS-CoV-2 antibody responses, support using the RBD antigen in serological diagnostic assays and RBD-specific antibody levels as a correlate of SARS-CoV-2 neutralizing antibodies in people.

Evaluation of nine commercial SARS-CoV-2 immunoassays

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.

Profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers.

A new coronavirus (severe acute respiratory syndrome coronavirus [SARS-CoV]) has been identified to be the etiological agent of severe acute respiratory syndrome. Given the highly contagious and acute nature of the disease, there is an urgent need for the development of diagnostic assays that can detect SARS-CoV infection. For determination of which of the viral proteins encoded by the SARS-CoV genome may be exploited as diagnostic antigens for serological assays, the viral proteins were expressed individually in mammalian and/or bacterial cells and tested for reactivity with sera from SARS-CoV-infected patients by Western blot analysis. A total of 81 sera, including 67 from convalescent patients and seven pairs from two time points of infection, were analyzed, and all showed immunoreactivity towards the nucleocapsid protein (N). Sera from some of the patients also showed immunoreactivity to U274 (59 of 81 [73%]), a protein that is unique to SARS-CoV. In addition, all of the convalescent-phase sera showed immunoreactivity to the spike (S) protein when analyzed by an immunofluorescence method utilizing mammalian cells stably expressing S. However, samples from the acute phase (2 to 9 days after the onset of illness) did not react with S, suggesting that antibodies to N may appear earlier than antibodies to S. Alternatively, this could be due to the difference in the sensitivities of the two methods. The immunoreactivities to these recombinant viral proteins are highly specific, as sera from 100 healthy donors did not react with any of them. These results suggest that recombinant N, S, and U274 proteins may be used as antigens for the development of serological assays for SARS-CoV.

Will antibody tests for the coronavirus really change everything?

Colonization of Severe Acute Respiratory Syndrome-Associated Coronavirus Among Health-Care Workers Screened by Nasopharyngeal Swab

Study objectives To report the efficacy and findings of a large-scale preventive screening program for severe acute respiratory syndrome-associated coronavirus (SARS-CoV) using amplification of the virus from a nasopharyngeal swab (NPS) obtained from the health-care workers (HCWs). Design A prospective observational study. Setting A medical center in Taiwan. Participants Two hundred thirty HCWs. Intervention NPS examination for the presence of SARS-CoV by two nested reverse transcription-polymerase chain reaction (RT-PCR) assays. Measurements and results During the outbreak of severe acute respiratory syndrome (SARS), NPS polymerase chain reaction screening of HCWs for SARS-CoV was performed. SARS-CoV was examined by two nested RT-PCRs and a quantitative RT-PCR. Serum-specific antibodies were assessed by enzyme immunoassay and indirect immunofluorescence. We monitored 230 HCWs, including 217 first-line HCWs and 13 non–first-line HCWs. One hundred ninety first-line HCWs and 13 non–first-line HCWs had negative results in both nested RT-PCR assays. Two first-line HCWs who were positive on both nested RT-PCR assays had SARS. They had 16,900 ± 7,920 copies (mean ± SD) of RNA per milliliter in the NPS and had detectable anti-SARS antibodies. The remaining 25 first-line HCWs were negative for the first nested RT-PCR but positive for the second nested RT-PCR. Their corresponding titers were 338 ± 227 copies of RNA per milliliter; antibodies developed in none of these 25 HCWs. The expression and function of angiotensin-converting enzyme-2 were not different among these HCWs. This study shows that colonization of SARS-CoV occurred in 25 of 217 well-protected first-line HCWs on a SARS-associated service, but they remained seronegative. Conclusion With the second RT-PCR assay more sensitive than the first RT-PCR assay, we are able to show that approximately 11.5% of well-protected HCWs exposed to SARS patients or specimens may have colonization without seroconversion. Only those with significant clinical symptoms or disease would have active immunity. Thus, regular NPS screening for nested RT-PCR assays in conjunction with a daily recording of body temperature in all first-line HCWs may provide an effective way of early detection.

Coronavirus vaccine developers wary of errant antibodies

Detection of feline coronavirus infection in southern African nondomestic felids.

Feline coronavirus (FCoV) infects members of the Felidae family with results ranging from seroconversion with no disease to fatal feline infectious peritonitis (FIP). Infection of non-domestic felids with FCoV is of concern, particularly in endangered populations such as cheetahs (Acinonyx jubatus). In this investigation, we tested 342 animals in the Republic of South Africa and Namibia, including 140 animals from wild populations, for evidence of FCoV infection by serology and/or reverse transcription/nested polymerase chain reaction (RT/nPCR) on feces from 1999 through 2001. Past or current infection was evaluated. Of these, 195 animals had evidence of infection and included 41 animals from wild populations. Serology (indirect immunofluorescence) did not always correlate with viral RNA detection, as seronegative animals were occasionally virus-positive, while many seropositive animals were not shedding virus. Serology indicated the infecting virus was most closely related to type I FCoV. Antibody levels in the majority of animals were low, even in those actively infected. Ten of 48 animals tested at more than one time point by RT/nPCR were shedding virus at multiple time points possibly indicating persistent infection. Infection in free-ranging animals was also notable, as over a quarter of the free-ranging animals tested had evidence of current or previous FCoV infection. Testing by serology and RT/nPCR is recommended for screening for FCoV infection.

Seroepidemiologic Study Designs for Determining SARS-COV-2 Transmission and Immunity

Serological signatures of SARS-CoV-2 infection: Implications for antibody-based diagnostics

Background The antibody response generated following infection with SARS-CoV-2 is expected to decline over time. This may cause individuals with confirmed SARS-CoV-2 infection to test negative according to serological diagnostic tests in the months and years following symptom onset. Methods A multiplex serological assay was developed to measure IgG and IgM antibody responses to four SARS-CoV-2 Spike (S) antigens: spike trimeric ectodomain (Stri), its receptor-binding domain (RBD), spike subunit 1 (S1), and spike subunit 2 (S2). Antibody responses were measured in serum samples from patients in French hospitals with RT-qPCR confirmed infection (n = 259), and negative control serum samples collected before the start of the SARS-CoV-2 epidemic in 2019 (n = 335). The multiplex antibody data was used to train a random forests algorithm for classifying individuals with previous SARS-CoV-2 infection. A mathematical model of antibody kinetics informed by prior information from other coronaviruses was used to estimate time-varying antibody responses and assess the potential sensitivity and classification performance of serological diagnostics during the first year following symptom onset. Results IgG antibody responses to one S antigen identified individuals with previous RT-qPCR confirmed SARS-CoV-2 infection with 90.3% sensitivity (95% confidence interval (CI); 86.1%, 93.4%) and 99.1% specificity (95% CI; 97.4%, 99.7%). Using a serological signature of IgG to four antigens, it was possible to identify infected individuals with 96.1% sensitivity (95% CI; 93.0%, 97.9%) and 99.1% specificity (95% CI; 97.4%, 99.7%). Antibody responses to SARS-CoV-2 increase rapidly 1-2 weeks after symptom onset, with antibody responses predicted to peak within 2-4 weeks. Informed by prior data from other coronaviruses, one year following symptom onset antibody responses are predicted to decay by approximately 60% from the peak response. Depending on the selection of sero-positivity cutoff, we estimate that the sensitivity of serological diagnostics may reduce to 56%-97% after six months, and to 49%-93% after one year. Conclusion Serological signatures based on antibody responses to multiple antigens can provide more accurate and robust serological classification of individuals with previous SARS-CoV-2 infection. Changes in antibody levels over time may cause reductions in the sensitivity of serological diagnostics leading to an underestimation of sero-prevalence. It is essential that data continue to be collected to evaluate this potential risk.

[Coronaviruses in pathology].

The crown-like projections on the surface of the particles permits differentiation of coronaviruses from other riboviruses and their individualization as a distinct family. Recognition of their role in the aetiology of respiratory, enteral and encephalitic infections has promoted investigations for the finding of preventive vaccines. Selection of the strains showed that their immunogenicity depends upon the density and salience of the crown-like projections, the antigenic relationship of the projections determining cross serologic reactions between different species. At present experiments are being carried out on animals with vaccines prepared with antigenically related heterologous cornavirus species, not pathogenic for the host, and with subunitary vaccines prepared from the projections of the homologous species. The Coronaviridae family established on morphologic criteria is thus confirmed by the immunologic relationship and specificity of its members.

Antibody Tests in Detecting SARS-CoV-2 Infection: A Meta-Analysis

The emergence of Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 made imperative the need for diagnostic tests that can identify the infection. Although Nucleic Acid Test (NAT) is considered to be the gold standard, serological tests based on antibodies could be very helpful. However, individual studies are usually inconclusive, thus, a comparison of different tests is needed. We performed a systematic review and meta-analysis in PubMed, medRxiv and bioRxiv. We used the bivariate method for meta-analysis of diagnostic tests pooling sensitivities and specificities. We evaluated IgM and IgG tests based on Enzyme-linked immunosorbent assay (ELISA), Chemiluminescence Enzyme Immunoassays (CLIA), Fluorescence Immunoassays (FIA), and the Lateral Flow Immunoassays (LFIA). We identified 38 studies containing data from 7848 individuals. Tests using the S antigen are more sensitive than N antigen-based tests. IgG tests perform better compared to IgM ones and show better sensitivity when the samples were taken longer after the onset of symptoms. Moreover, a combined IgG/IgM test seems to be a better choice in terms of sensitivity than measuring either antibody alone. All methods yield high specificity with some of them (ELISA and LFIA) reaching levels around 99%. ELISA- and CLIA-based methods perform better in terms of sensitivity (90%–94%) followed by LFIA and FIA with sensitivities ranging from 80% to 89%. ELISA tests could be a safer choice at this stage of the pandemic. LFIA tests are more attractive for large seroprevalence studies but show lower sensitivity, and this should be taken into account when designing and performing seroprevalence studies.

Nasopharyngeal and Oropharyngeal Swabs, And/Or Serology for SARS COVID-19: What Are We Looking For?

Governments and clinicians that were fully involved in the dramatic SARS-CoV-2 outbreak during the last few weeks in Italy (and more or less all over the world) are fiercely debating the use of methods for screening this viral infection. Thus, all countries are employing a lot of resources in order to test more and more subjects. For this purpose, there are different strategies, based on either direct or indirect tests. Among the first category, the main assays used for SARS-CoV-2 are based on a real-time reverse transcriptase polymerase chain reaction (RT-PCR). Such tests can be performed on nasopharyngeal and oropharyngeal swabs for the categories of those with symptoms and those potentially exposed. In order to integrate the molecular assays in the diagnosis of SARS-CoV-2, a wide range of serology immunoassays (IAs) have also been developed. If we want to identify “immune” people in order to let them to come back to work, serology is the best (and probably the only) approach.

SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup

In late 2019, cases of atypical pneumonia were detected in China. The etiological agent was quickly identified as a betacoronavirus (named SARS-CoV-2), which has since caused a pandemic. Several methods allowing for the specific detection of viral nucleic acids have been established, but these only allow detection of the virus during a short period of time, generally during acute infection. Serological assays are urgently needed to conduct serosurveys, to understand the antibody responses mounted in response to the virus, and to identify individuals who are potentially immune to re-infection. Here we describe a detailed protocol for expression of antigens derived from the spike protein of SARS-CoV-2 that can serve as a substrate for immunological assays, as well as a two-stage serological enzyme-linked immunosorbent assay (ELISA). These assays can be used for research studies and for testing in clinical laboratories. © 2020 The Authors. Basic Protocol 1: Mammalian cell transfection and protein purification Basic Protocol 2: A two-stage ELISA for high-throughput screening of human serum samples for antibodies binding to the spike protein of SARS-CoV-2.

Establishment of serological test to detect antibody against ferret coronavirus

Since there is no available serological methods to detect antibodies to ferret coronavirus (FRCoV), an enzyme-linked immunosorbent assay (ELISA) using recombinant partial nucleocapsid (N) proteins of the ferret coronavirus (FRCoV) Yamaguchi-1 strain was developed to establish a serological method for detection of FRCoV infection. Many serum samples collected from ferrets recognized both a.a. 1–179 and a.a. 180–374 of the N protein, but two serum samples did not a.a. 180–374 of the N protein. This different reactivity was also confirmed by immunoblot analysis using the serum from the ferret.Therefore, the a.a. 1–179 of the N protein was used as an ELISA antigen. Serological test was carried out using sera or plasma of ferrets in Japan. Surprisingly, 89% ferrets in Japan had been infected with FRCoV. These results indicated that our established ELISA using a.a. 1–179 of the N protein is useful for detection of antibody to FRCoV for diagnosis and seroepidemiology of FRCoV infection.

Isolation of coronaviruses antigenically indistinguishable from bovine coronavirus from wild ruminants with diarrhea.

Diarrheal feces from three sambar deer and one waterbuck in a wild animal habitat and one white-tailed deer on a wildlife farm in Ohio contained coronavirus particles which were agglutinated by antiserum to bovine coronavirus (BCV) in immune electron microscopy. Three coronavirus strains were isolated in human rectal tumor cells from the feces of the sambar and white-tailed deer and the waterbuck, respectively. Hemagglutination, receptor-destroying enzyme activity, indirect immunofluorescence, hemagglutination inhibition, virus neutralization, and Western blot (immunoblot) tests showed close biological and antigenic relationships among the isolates and with selected BCV strains. Gnotobiotic and colostrum-deprived calves inoculated with each of these isolates developed diarrhea and shed coronavirus in their feces and from their nasal passages. In a serological survey of coronavirus infections among wild deer, 8.7 and 6.6% of sera from mule deer in Wyoming and from white-tailed deer in Ohio, respectively, were seropositive against both of the isolates and selected BCV isolates by indirect immunofluorescence tests. These results confirm the existence of coronaviruses in wild ruminants and suggest that these species may harbor coronavirus strains transmissible to cattle.

Coronavirus antibodies in sera from patients with multiple sclerosis and matched controls.

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.

The Role of Antibody Testing for SARS-CoV-2: Is There One?

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) brought with it rapid development of both molecular and serologic assays for identification of COVID-19 infections. While Food and Drug Administration (FDA) emergency use authorization (EUA) is required for clinical application of SARS-CoV-2 molecular tests, submission for EUA is currently a voluntary process for manufacturers of serologic assays. The absence of FDA oversight of serologic tests is concerning, given that the commercially available serologic assays are highly variable, differing in their format, the antibody class detected, the targeted antigen and the acceptable specimen types. An added complication is the lack of a clear understanding for how such assays should be utilized and what the reported results ultimately indicate, or perhaps more importantly, what they do not indicate. Here, we provide a brief summary of the performance of a number of serologic assays reported in the literature, comment on what we do and do not know regarding our immune response to SARS-CoV-2, and provide a number of scenarios for which serologic testing will play a role in during our global response to this pandemic.

An Outbreak of Human Coronavirus OC43 Infection and Serological Cross-reactivity with SARS Coronavirus.

BACKGROUND In summer 2003, a respiratory outbreak was investigated in British Columbia, during which nucleic acid tests and serology unexpectedly indicated reactivity for severe acute respiratory syndrome coronavirus (SARS-CoV). METHODS Cases at a care facility were epidemiologically characterized and sequentially investigated for conventional agents of respiratory infection, SARS-CoV and other human CoVs. Serological cross-reactivity between SARS-CoV and human CoV-OC43 (HCoV-OC43) was investigated by peptide spot assay. RESULTS Ninety-five of 142 residents (67%) and 53 of 160 staff members (33%) experienced symptoms of respiratory infection. Symptomatic residents experienced cough (66%), fever (21%) and pneumonia (12%). Eight residents died, six with pneumonia. No staff members developed pneumonia. Findings on reverse transcriptase-polymerase chain reaction assays for SARS-CoV at a national reference laboratory were suspected to represent false positives, but this was confounded by concurrent identification of antibody to N protein on serology. Subsequent testing by reverse transcriptase-polymerase chain reaction confirmed HCoV-OC43 infection. Convalescent serology ruled out SARS. Notably, sera demonstrated cross-reactivity against nucleocapsid peptide sequences common to HCoV-OC43 and SARS-CoV. CONCLUSIONS These findings underscore the virulence of human CoV-OC43 in elderly populations and confirm that cross-reactivity to antibody against nucleocapsid proteins from these viruses must be considered when interpreting serological tests for SARS-CoV.

SARS Antibody Test for Serosurveillance

A peptide-based enzyme-linked immunosorbent assay (ELISA) can be used for retrospective serosurveillance of severe acute respiratory syndrome (SARS) by helping identify undetected chains of disease transmission. The assay was developed by epitope mapping, using synthetic peptides from the spike, membrane, and nucleocapsid protein sequences of SARS-associated coronavirus. The new peptide ELISA consistently detected seroconversion by week 2 of onset of fever, and seropositivity remained through day 100. Specificity was 100% on normal blood donor samples, on serum samples associated with infection by other pathogens, and on an interference panel. The peptide-based test has advantages of safety, standardization, and automation over previous immunoassays for SARS. The assay was used for a retrospective survey of healthy healthcare workers in Taiwan who treated SARS patients. Asymptomatic seroconversions were detected in two hospitals that had nosocomial disease.

Virus neutralization study using H120, H52, 793/B antisera against Iranian infectious bronchitis virus genotypes.

Infectious bronchitis virus (IBV), a major pathogen of the domestic fowl, exhibits extensive antigenic variation. IBV is a member of the Coronaviridae family and the genus Gammacoronavirus. A new infectious bronchitis virus serotype can emerge from only very few amino acid changes within the major peplomer glycoprotein, namely in its S1 part forming the virion spike. Principally, the serotypes are identified by virus neutralization (VN) tests. This study is aimed to investigate the neutralizing efficiency of H52, H120, and 4/91 antiserum against IBV genotypes (IS-1494, IS-720, 793/B, IR-1) recently circulating in Iran. For the first time, we have used cross-neutralization tests for the serological classification of these isolates. In this study, all antisera failed to neutralize all IBV strains. According to the results of our research, cross-protection studies are necessary for the design of a proper vaccination program for IBV circulating genotypes in Iran. The data are useful for the development of new vaccine strategies. Keywords: avian infectious bronchitis; Iran; virus neutralization.

Reliability of serological tests for COVID-19: Comparison of three immunochromatography test kits for SARS-CoV-2 antibodies.

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.

Determination of coronavirus 229E antibody by an immune‐adherence hemagglutination method

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.

Detection of a coronavirus from turkey poults in Europe genetically related to infectious bronchitis virus of chickens.

Intestinal contents of 13-day-old turkey poults in Great Britain were analysed as the birds showed stunting, unevenness and lameness, with 4% mortality. At post mortem examination, the main gross features were fluid caecal and intestinal contents. Histological examination of tissues was largely unremarkable, apart from some sections that showed crypt dilation and flattened epithelia. Negative contrast electron microscopy of caecal contents revealed virus particles, which in size and morphology had the appearance of a coronavirus. RNA was extracted (turkey/UK/412/00) and used in a number of reverse transcription-polymerase chain reactions (RT-PCRs) with the oligonucleotides based on sequences derived from avian infectious bronchitis virus (IBV), a coronavirus of domestic fowl. The RT-PCRs confirmed that turkey/UK/412/00 was a coronavirus and, moreover, showed that it had the same partial gene order (S-E-M-5-N-3' untranslated region) as IBV. This gene order is unlike that of any known mammalian coronavirus, which does not have a gene analogous to the gene 5 of IBV.The gene 5 of the turkey virus had two open reading frames, 5a and 5b, as in IBV and the coronaviruses isolated from turkeys in North America. The turkey/UK/412/00 also resembled IBV, but not mammalian coronaviruses, in having three open reading frames in the gene encoding E protein (gene 3). The percentage differences between the nucleotide sequences of genes 3 and 5 and the 3' untranslated region of turkey/UK/412/00 when compared with those of IBVs were similar to the differences observed when different strains of IBV were compared with each other. No sequences unique to the turkey isolates were identified. These results demonstrate, for the first time, that a coronavirus was associated with disease in turkeys outside of North America and that it is a Group 3 coronavirus, like IBV.

SARS-CoV-2 infection serology: a useful tool to overcome lockdown?

The outbreak of 2019 novel coronavirus disease (Covid-19) caused by SARS-CoV-2 has spread rapidly, inducing a progressive growth in infected patients number. Social isolation (lockdown) has been assessed to prevent and control virus diffusion, leading to a worldwide financial and political crisis. Currently, SARS-CoV-2 RNA detection in nasopharyngeal swab takes place by real-time PCR (RT-qPCR). However, molecular tests can give some false-negative results. In this context, serological assays can be useful to detect IgG/IgM antibodies, to assess the degree of immunization, to trace the contacts, and to support the decision to re-admit people at work. A lot of serological diagnostic kits have been proposed on the market but validation studies have not been published for many of them. The aim of our work was to compare and to evaluate different assays analytical performances (two different immunochromatographic cards, an immunofluorescence chromatographic card, and a chemiluminescence-automated immunoassay) on 43 positive samples with RT-qPCR-confirmed SARS-CoV-2 infection and 40 negative control subjects. Our data display excellent IgG/IgM specificities for all the immunocromatographic card tests (100% IgG and 100% IgM) and for the chemiluminescence-automated assay (100% IgG and 94% IgM); IgG/IgM sensitivities are moderately lower for all methods, probably due to the assay viral antigen's nature and/or to the detection time of nasopharyngeal swab RT-qPCR, with respect to symptoms onset. Given that sensitivities (around 94% and 84% for IgG and IgM, respectively) implicate false-negative cases and given the lack of effective vaccines or treatments, the only currently available procedure to reduce SARS-CoV-2 transmission is to identify and isolate persons who are contagious. For this reason, we would like to submit a flowchart in which serological tests, integrated with nasopharyngeal swab RT-qPCR, are included to help social and work activities implementation after the pandemic acute phase and to overcome lockdown.

Simultaneous detection of antibodies to mouse hepatitis virus recombinant structural proteins by a microsphere-based multiplex fluorescence immunoassay.

We describe a new microsphere-based multiplex fluorescent immunoassay (MFI) using recombinant mouse hepatitis virus (MHV) proteins to detect antibodies to coronaviruses in mouse and rat sera. All the recombinant proteins, including nucleocapsid (N) and 3 subunits of spike protein, S1, S2, and Smid, showed positive reactivity in MFI with mouse antisera to 4 MHV strains (MHV-S, -A59, -JHM, and -Nu67) and rat antiserum to a strain of sialodacryoadenitis virus (SDAV-681). The MFI was evaluated for its diagnostic power, with panels of mouse sera classified as positive or negative for anti-MHV antibodies by enzyme-linked immunosorbent assay (ELISA) using MHV virion antigen and indirect fluorescent antibody assay. The reactivities of 236 naturally infected mouse sera were examined; 227 samples were positive by MFI using S2 antigen (96% sensitivity), and 208 samples were positive using N antigen (88% sensitivity). Based on the assessment by MFI using the S2 and N antigens, only 3 serum samples showed double-negative results, indicating a false-negative rate of 1.3%. In 126 uninfected mouse sera, including 34 ELISA false-positive sera, only 7 samples showed false-positive results by MFI using either the S2 or N antigen (94% specificity). Similarly, the S2 and N antigen-based MFI was 98% sensitive and 100% specific in detecting anticoronavirus antibodies in rat sera. Thus, this MFI-based serologic assay using the S2 and N antigens promises to be a reliable diagnostic method, representing a highly sensitive and specific alternative to traditional ELISA for detection of coronavirus infections in laboratory mouse and rat colonies.

Samples with high virus load cause a trend toward lower signal in feline coronavirus antibody tests.

Measurement of feline coronavirus (FCoV) antibody titres is utilised mainly for diagnosing feline infectious peritonitis (FIP) and for quarantine purposes. However, occasional samples show a falsely low or negative FCoV antibody test. We tested the hypothesis that such results are due to virus in the sample binding antibody and rendering it unavailable to antigen in the test. Thirteen effusions, one plasma and three undefined samples from cats with FIP, which gave unexpectedly low FCoV antibody titres, were examined by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Increasing amounts of virus correlated with lower signals in indirect immunoflourescent, enzyme-linked immunosorbent asssay and rapid immunomigration antibody tests. However, five samples were negative by RT-PCR, so the presence of virus alone may not explain all cases of false-negative FCoV antibody tests, although it is a possible explanation in 71% of discordant samples. We conclude that falsely low or negative FCoV antibody tests can occur in samples rich in virus.

The spectrum of severe acute respiratory syndrome-associated coronavirus infection.

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.

Diagnostic accuracy of an automated chemiluminescent immunoassay for anti‐SARS‐CoV‐2 IgM and IgG antibodies: an Italian experience

A pandemic of coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has been spreading throughout the world. Though molecular diagnostic tests are the gold standard for COVID‐19, serological testing is emerging as a potential surveillance tool, in addition to its complementary role in COVID‐19 diagnostics. Indubitably quantitative serological testing provides greater advantages than qualitative tests but today there is still little known about serological diagnostics and what the most appropriate role quantitative tests might play. Sixty‐one COVID‐19 patients and 64 patients from a control group were tested by iFlash1800 CLIA analyzer for anti‐SARS CoV‐2 antibodies IgM and IgG. All COVID‐19 patients were hospitalized in San Giovanni di Dio Hospital (Florence, Italy) and had a positive oro/nasopharyngeal swab reverse‐transcription polymerase chain reaction result. The highest sensitivity with a very good specificity performance was reached at a cutoff value of 10.0 AU/mL for IgM and of 7.1 for IgG antibodies, hence near to the manufacturer's cutoff values of 10 AU/mL for both isotypes. The receiver operating characteristic curves showed area under the curve values of 0.918 and 0.980 for anti‐SARS CoV‐2 antibodies IgM and IgG, respectively. iFlash1800 CLIA analyzer has shown highly accurate results for the anti‐SARS‐CoV‐2 antibodies profile and can be considered an excellent tool for COVID‐19 diagnostics.

Antigenic Relationship between Human Coronavirus Strain DC 43 and Hemagglutinating Encephalomyelitis Virus Strain 67N of Swine: Antibody Responses in Human and Animal Sera

Hemagglutinating encephalomyelitis virus of swine (HEV) was adapted to growth in suckling mouse brain. Electron micrographs of HEV-infected suckling mouse brain, prepared by negative staining and thin-section techniques, exhibited typical morphological characteristics shared with other members of the Coronaviridae. The adaptation of HEV to suckling mouse brain facilitated serologic testing by the use of common host reagents and compatible animal systems. With hemagglutination inhibition, complement-fixation, and neutralization tests, an antigenic relationship was demonstrated between human coronavirus OC 43 and HEV in specific immune and hyperimmune animal sera. Children and adults with seroconversion to OC 43 antigen had diagnostic rises in titer of antibody to HEV antigens. Individuals with seroconversion to human coronaviruses 229E and B814 demonstrated antibody to HEV but not diagnostic rises in titer. Swine with titers of antibody to HEV had lower or no detectable titers of antibody to coronavirus OC 43. Although the prevalence and geometric mean titer of antibody to DC 43 were higher than the titer of antibody to HEV in every group of normal humans tested, significant differences in antibody response to coronavirus DC 43 and HEV were seen between populations that did or did not have possible contact with swine. The evidence suggested that antibody to HEV in humans probably represented a heterologous response to infection with coronavirus DC 43. However, a heterotypic response to unknown or uncharacterized strains of coronavirus cannot be excluded.

Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019

BACKGROUND: The novel coronavirus SARS-CoV-2 is a newly emerging virus. The antibody response in infected patient remains largely unknown, and the clinical values of antibody testing have not been fully demonstrated. METHODS: A total of 173 patients with SARS-CoV-2 infection were enrolled. Their serial plasma samples (n=535) collected during the hospitalization were tested for total antibodies (Ab), IgM and IgG against SARS-CoV-2. The dynamics of antibodies with the disease progress was analyzed. RESULTS: Among 173 patients, the seroconversion rate for Ab, IgM and IgG was 93.1%, 82.7% and 64.7%, respectively. The reason for the negative antibody findings in 12 patients might due to the lack of blood samples at the later stage of illness. The median seroconversion time for Ab, IgM and then IgG were day-11, day-12 and day-14, separately. The presence of antibodies was <40% among patients within 1-week since onset, and rapidly increased to 100.0% (Ab), 94.3% (IgM) and 79.8% (IgG) since day-15 after onset. In contrast, RNA detectability decreased from 66.7% (58/87) in samples collected before day-7 to 45.5% (25/55) during day 15-39. Combining RNA and antibody detections significantly improved the sensitivity of pathogenic diagnosis for COVID-19 (p<0.001), even in early phase of 1-week since onset (p=0.007). Moreover, a higher titer of Ab was independently associated with a worse clinical classification (p=0.006). CONCLUSIONS: The antibody detection offers vital clinical information during the course of SARS-CoV-2 infection. The findings provide strong empirical support for the routine application of serological testing in the diagnosis and management of COVID-19 patients.

Prevalence of antibody to human coronaviruses 229E, OC43 and neonatal calf diarrhea coronavirus (NCDCV) in patients of Northern Italy

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.

Establishment of a reference panel for the detection of anti-SARS-CoV antibodies

Abstract The immunological assays for detection of antibodies against SARS-CoV were developed in-house and some of them are available commercially. However, the antigens used in these assays differed. In order to validate the reliability of these assays, the standard panel should be established. In this study, we have expressed and purified severe acute respiratory syndrome (SARS) structural proteins and their fragments and developed indirect enzyme-linked immunosorbent assays (ELISAs) that detect antibodies against the SARS N, N1, N2, S1, SC, S2, and M proteins as well as the human coronavirus OC43 and 229E N proteins. These assays were used to screen 58 samples from SARS convalescent patients, 40 serial serum specimens from patients at different phases of SARS infection, and 88 plasma specimens from normal blood donors. The samples from normal blood donors were also tested for antibodies against other respiratory virus. The representative samples were chosen to comprise a reference panel of SARS antibodies that may be used for the detection of SARS. The panel is composed of 25 positive samples, 25 negative samples, 7 diluted samples for anti-N antibody, 6 diluted samples for anti-S antibody, and one sample for validating precision. Comparison of detection results with different SARS antibody assays indicated that our panel should differentiate the specificity and sensitivity of different assays.

Connecting clusters of COVID-19: an epidemiological and serological investigation

Summary Background Elucidation of the chain of disease transmission and identification of the source of coronavirus disease 2019 (COVID-19) infections are crucial for effective disease containment. We describe an epidemiological investigation that, with use of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays, established links between three clusters of COVID-19. Methods In Singapore, active case-finding and contact tracing were undertaken for all COVID-19 cases. Diagnosis for acute disease was confirmed with RT-PCR testing. When epidemiological information suggested that people might have been nodes of disease transmission but had recovered from illness, SARS-CoV-2 IgG serology testing was used to establish past infection. Findings Three clusters of COVID-19, comprising 28 locally transmitted cases, were identified in Singapore; these clusters were from two churches (Church A and Church B) and a family gathering. The clusters in Church A and Church B were linked by an individual from Church A (A2), who transmitted SARS-CoV-2 infection to the primary case from Church B (F1) at a family gathering they both attended on Jan 25, 2020. All cases were confirmed by RT-PCR testing because they had active disease, except for A2, who at the time of testing had recovered from their illness and tested negative. This individual was eventually diagnosed with past infection by serological testing. ELISA assays showed an optical density of more than 1·4 for SARS-CoV-2 nucleoprotein and receptor binding domain antigens in titres up to 1/400, and viral neutralisation was noted in titres up to 1/320. Interpretation Development and application of a serological assay has helped to establish connections between COVID-19 clusters in Singapore. Serological testing can have a crucial role in identifying convalescent cases or people with milder disease who might have been missed by other surveillance methods. Funding National Research Foundation (Singapore), National Natural Science Foundation (China), and National Medical Research Council (Singapore).

The RBD Of The Spike Protein Of SARS-Group Coronaviruses Is A Highly Specific Target Of SARS-CoV-2 Antibodies But Not Other Pathogenic Human and Animal Coronavirus Antibodies

A new Severe Acute Respiratory Syndrome Coronavirus variant (SARS-CoV-2) that first emerged in late 2019 is responsible for a pandemic of severe respiratory illness. People infected with this highly contagious virus present with clinically inapparent, mild or severe disease. Currently, the presence of the virus in individual patients and at the population level is being monitored by testing symptomatic cases by PCR for the presence of viral RNA. There is an urgent need for SARS-CoV-2 serologic tests to identify all infected individuals, irrespective of clinical symptoms, to conduct surveillance and implement strategies to contain spread. As the receptor binding domain (RBD) of the viral spike (S) protein is poorly conserved between SARS-CoVs and other pathogenic human coronaviruses, the RBD represents a promising antigen for detecting CoV specific antibodies in people. Here we use a large panel of human sera (70 SARS-CoV-2 patients and 71 control subjects) and hyperimmune sera from animals exposed to zoonotic CoVs to evaluate the performance of the RBD as an antigen for accurate detection of SARS-CoV-2-specific antibodies. By day 9 after the onset of symptoms, the recombinant SARS-CoV-2 RBD antigen was highly sensitive (98%) and specific (100%) to antibodies induced by SARS-CoVs. We observed a robust correlation between levels of RBD binding antibodies and SARS-CoV-2 neutralizing antibodies in patients. Our results, which reveal the early kinetics of SARS-CoV-2 antibody responses, strongly support the use of RBD-based antibody assays for population-level surveillance and as a correlate of neutralizing antibody levels in people who have recovered from SARS-CoV-2 infections.

Novel surrogate virus neutralization test reveals low serum neutralizing anti-SARS-CoV-2-S antibodies levels in mildly affected COVID-19 convalescents

Neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into cells using surface-expressed angiotensin-converting enzyme 2 (ACE2). We developed a surrogate neutralization test (sVNT) to assess at what degree serum antibodies interfere with the binding of SARS-CoV-2-S-RBD to ACE2. The sVNT revealed neutralizing anti-SARS-CoV-2-S antibodies in the sera of 90% of mildly and 100% of severely affected coronavirus-disease-2019 (COVID-19) convalescent patients. Importantly, sVNT results correlated strongly to the results from pseudotyped-vesicular stomatitis virus-vector-based neutralization assay and to levels of anti-SARS-CoV-2-S1 IgG and IgA antibodies. Moreover, levels of neutralizing antibodies also correlated to duration and severity of clinical symptoms, but not patient age or gender. These findings together with the sVNT will not only be important for evaluating the prevalence of neutralizing antibodies in a population but also for identifying promising plasma donors for successful passive antibody therapy.

Experimental inoculation of cats with human coronavirus 229E and subsequent challenge with feline infectious peritonitis virus.

Minimal-disease cats exposed to live human coronavirus 229E developed homologous antibody responses that suggested little or no replication of the virus in inoculated animals. Oronasal and subcutaneous inoculation of coronavirus 229E did not elicit an antibody response by heterologous (transmissible gastroenteritis virus, canine coronavirus) neutralization or by heterologous (transmissible gastroenteritis virus) kinetics-based enzyme-linked immunosorbent assay. No clinical signs attributable to coronavirus 229E were seen in inoculated cats. Although the number of animals in each of the five experimental groups was small (n = 2), antibodies produced in response to the virus did not appear to sensitize cats to subsequent feline infectious peritonitis virus challenge, but neither did they cross-protect cats against the challenge dose.

Diagnostic methods and potential portable biosensors for coronavirus disease 2019

Timely detection and diagnosis are urgently needed to guide epidemiological measures, infection control, antiviral treatment, and vaccine research. In this review, biomarkers/indicators for diagnosis of coronavirus disease 2019 (COVID-19) or detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the environment are summarized and discussed. It is concluded that the detection methods targeting antibodies are not suitable for screening of early and asymptomatic cases since most patients had an antibody response at about 10 days after onset of symptoms. However, antibody detection methods can be combined with quantitative real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) to significantly improve the sensitivity and specificity of diagnosis, and boost vaccine research. Fast, sensitive and accurate detection methods targeting antigens need to be developed urgently. Various specimens for diagnosis or detection are compared and analyzed. Among them, deep throat saliva and induced sputum are desired for RT-qPCR test or other early detection technologies. Chest computerized tomography (CT) scan, RT-qPCR, lateral flow immunochromatographic strip (LFICS) for diagnosis of COVID-19 are summarized and compared. Specially, potential electrochemical (EC) biosensor, surface enhanced Raman scattering (SERS)-based biosensor, field-effect transistor (FET)-based biosensor, surface plasmon resonance (SPR)-based biosensor and artificial intelligence (AI) assisted diagnosis of COVID-19 are emphasized. Finally, some commercialized portable detection device, current challenges and future directions are discussed.

The outbreak of 2019 novel coronavirus disease (Covid-19) caused by SARS-CoV-2 has spread rapidly, inducing a progressive growth in infected patients number. Social isolation (lockdown) has been assessed to prevent and control virus diffusion, leading to a worldwide financial and political crisis. Currently, SARS-CoV-2 RNA detection in nasopharyngeal swab takes place by real-time PCR (RT-qPCR). However, molecular tests can give some false-negative results. In this context, serological assays can be useful to detect IgG/IgM antibodies, to assess the degree of immunization, to trace the contacts, and to support the decision to re-admit people at work. A lot of serological diagnostic kits have been proposed on the market but validation studies have not been published for many of them. The aim of our work was to compare and to evaluate different assays analytical performances (two different immunochromatographic cards, an immunofluorescence chromatographic card, and a chemiluminescence-automated immunoassay) on 43 positive samples with RT-qPCR-confirmed SARS-CoV-2 infection and 40 negative control subjects. Our data display excellent IgG/IgM specificities for all the immunocromatographic card tests (100% IgG and 100% IgM) and for the chemiluminescence-automated assay (100% IgG and 94% IgM); IgG/IgM sensitivities are moderately lower for all methods, probably due to the assay viral antigen’s nature and/or to the detection time of nasopharyngeal swab RT-qPCR, with respect to symptoms onset. Given that sensitivities (around 94% and 84% for IgG and IgM, respectively) implicate false-negative cases and given the lack of effective vaccines or treatments, the only currently available procedure to reduce SARS-CoV-2 transmission is to identify and isolate persons who are contagious. For this reason, we would like to submit a flowchart in which serological tests, integrated with nasopharyngeal swab RT-qPCR, are included to help social and work activities implementation after the pandemic acute phase and to overcome lockdown.

Coronaviral hypothetical and structural proteins were found in the intestinal surface enterocytes and pneumocytes of severe acute respiratory syndrome (SARS)

Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease that haunted the world from November 2002 to July 2003. Little is known about the biology and pathophysiology of the novel coronavirus that causes SARS. The tissue and cellular distributions of coronaviral hypothetical and structural proteins in SARS were investigated. Antibodies against the hypothetical (SARS 3a, 3b, 6, 7a and 9b) and structural proteins (envelope, membrane, nucleocapsid and spike) of the coronavirus were generated from predicted antigenic epitopes of each protein. The presence of these proteins were first verified in coronavirus-infected Vero E6 tissue culture model. Immunohistochemical studies on different human tissues, including a cohort of nine autopsies, two liver biopsies and intestinal biopsies of SARS patients, further confirmed the existence of coronaviral hypothetical and structural proteins in the cytoplasm of pneumocytes and small intestinal surface enterocytes in SARS patients. With this vast array of antibodies, no signal was observed in other cell types including those organs in which reverse transcriptase-polymerase chain reactions were reported to be positive. Structural proteins and the functionally undefined hypothetical proteins were expressed in coronavirus-infected cells with distinct expression pattern in different organs in SARS patients. These antipeptide antibodies can be useful for the diagnosis of SARS at the tissue level.

Serological relationships of the subcomponents of human coronavirus strain 229E and mouse hepatitis virus strain 3.

Antibodies were raised in rabbits against the structural components of human coronavirus strain 229E and mouse hepatitis virus strain 3, prepared from disrupted virus particles. Hyperimmune sera to the subcomponents showed cross-reactions by enzyme-linked immunosorbent assay between ribonucleoprotein antigens of these viruses, indicating the presence of a common antigen(s). None of the other virus structural components showed any cross-reactivity.

Detection of turkey enteric coronavirus by enzyme-linked immunosorbent assay and differentiation from other coronaviruses.

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.

Evaluations of serological test in the diagnosis of 2019 novel coronavirus (SARS-CoV-2) infections during the COVID-19 outbreak

The ongoing SARS-CoV-2 outbreak has killed over twenty-one thousand and sickened over four hundred thousand people worldwide, posing a great challenge to global public health. A sensitive and accurate diagnosis method will substantially help to control disease expansion. Here, we developed a chemiluminescence-immunoassay method based on the recombinant nucleocapsid antigen and the magnetic beads for diagnosis of SARS-CoV-2 infections and surveillance of antibody changing pattern. Serums from 29 healthy individuals, 51 tuberculosis patients, and 79 SARS-CoV-2 confirmed patients were employed to evaluate the performance of this approach. Compared to the IgM testing, the IgG testing was more reliable in which it identified 65 SARS-CoV-2 infections from the 79 confirmed patients and only two false-positive cases from the 80 control group with a sensitivity and specificity reaching 82.28% and 97.5%, respectively. However, only a slight difference (not statistically significant) in the detected cases of SARS-CoV-2 infections was observed between the IgM and IgG testing manner in patients at a different time of onset of disease. A performance comparison between an ELISA kit using the same nucleocapsid antigen and our chemiluminescence method was undertaken. The same false-positive cases were seen in both methods from the paired control group, while ELISA kit can only detect half of the SARS-CoV-2 infections from paired SARS-CoV-2 confirmed patients group than that of the chemiluminescence method, indicating a higher performance for the chemiluminescence-immunoassay approach. Together, our studies provide a useful and valuable serological testing tool for the diagnosis of SARS-CoV-2 infections in the community.

Detection of human coronavirus 229E-specific antibodies using recombinant fusion proteins

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.