PMC:7572153 / 23710-29561 JSONTXT

{"project":"MyTest","denotations":[{"id":"33078221-32284614-28920603","span":{"begin":1507,"end":1509},"obj":"32284614"},{"id":"33078221-32267220-28920604","span":{"begin":1812,"end":1814},"obj":"32267220"},{"id":"33078221-32267220-28920605","span":{"begin":2842,"end":2844},"obj":"32267220"},{"id":"33078221-20943876-28920606","span":{"begin":3239,"end":3241},"obj":"20943876"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"text":"Discussion\nWhile rigorous measures led to a partial control of the recent SARS-CoV-2 pandemic in some countries, validated serological assays are needed to consolidate those achievements and to support the transition to a post-peak phase. This includes for example diagnostic measures for late/post-infection stages, COVID-19 contact tracing, the assessment of epidemiological aspects, and the evaluation of immunity after infection or in potential vaccine trials. In the recent study, we validated an in-house flow cytometric assay for the detection of SARS-CoV-2S-specific IgM and IgG using sera from PCR-confirmed COVID-19 cases and a collection of control serum samples. In regard to specificity and sensitivity, our flow cytometric assay showed a comparable or even better performance compared to commercial CE-marked serological assays (EuroImmun ELISA and Shenzhen Yhlo CLIA) (Table 1).\nDetection of viral nucleic acids via real-time PCR is the gold standard in the diagnosis of acute SARS-CoV-2 infections. However, despite its reliability early during infection, confirmation of an infection at later time points becomes less reliable. As early as 8 days post-infection, the diagnostic value of serological assays might therefore outperform nucleic acid–based methods [20, 21]. Indeed, also our study showed seroconversion in a longitudinal set of sera from one patient 8 days after the first positive PCR test, although the exact infection date is not clearly defined. As reported before [20, 22, 23], IgM did not generally possess a higher clinical sensitivity compared to IgG, since most of the IgM+ specimen tested in the present study were positive for both isotypes. Detection of SARS-CoV-2-specific IgA was previously reported as more sensitive than detection of IgG in the EuroImmun ELISA kits [14]. However, while this held also true in our study, IgG and IgM measured by the flow cytometric assay were similarly sensitive compared to the IgA ELISA. This higher sensitivity to detect S-specific IgG might be due to the different viral antigens used in the assays. Our flow cytometric assay exploits full-length S protein in its natural conformation and with the respective post-translational modifications due to the expression in mammalian cells. This enables detection of the full spectrum of S-specific antibodies directed against conformational epitopes and glycosylated sites as well, some immunogenic sites possibly missing in truncated, recombinant S1-only proteins as used in the EuroImmun ELISA.\nA potential downside of using full-length S for serological testing might be the detection of cross-reactive antibodies induced by other HCoV. Along this line, some assays detect only antibodies directed against the S1 subunit (like the EuroImmun ELISA) or the receptor-binding domain in order to increase specificity [14, 15]. However, in a collection of sera from individuals that suffered from an infection with an endemic HCoV shortly before blood collection, none was tested positive for SARS-CoV-2 antibodies. In additional 180 specimens sampled before the COVID-19 outbreak, two sera were found to be reactive in the flow cytometric assay. Since endemic HCoV seroprevalence is high in the general population [11] and those two individuals were non-reactive in the commercial S1-specific ELISA, a plausible explanation is cross-reaction of antibodies induced by the endemic HCoVs with the S2 subunit of SARS-CoV-2. Although the reactivity of the two specimens needs to be classified as false-positive detection of SARS-CoV-2 antibodies, these cross-reactive antibodies might possess antiviral activity against COVID-19 and the analysis of cross-protection due to these responses might be an interesting topic for further investigations.\nRegarding the clinical sensitivity, the flow cytometric serology assay detected 100% of IgM- and IgG-positive samples measured by either of the two commercial assays. Only in cases where blood samples were taken at the same day as PCR sampling, all assays (ELISA, CLIA, cytometry) were negative, probably reflecting acute infections prior to development of detectable antibody responses. The lower analytical detection limit of the flow cytometric assay is consistent with its excellent clinical sensitivity.\nSince in the early phase of such a pandemic, there are naturally a limited number of reliable serological test kits available, and there is a high need to expand the portfolio of serology techniques which can be rapidly applied and scaled up. The flow cytometry–based technique to detect SARS-CoV-2 seroconversion presented here fulfills fundamental criteria in regard to sensitivity, specificity, and robustness. The basic requirements needed like cell culture, plastic ware, and a flow cytometer are available in many standard diagnostic and biomedical research labs. Although performing the assay and analyzing the primary data need some trained personal, high-throughput solutions of this method can increase serology testing capacities significantly without competing for ELISA/CLIA kits. Given the large number of antibody assays reaching the market without clearly defined analytical sensitivities, using recombinant ACE-2 Fc protein for standardization is a potential strategy for cross-assay comparisons. Moreover, the quantification of S-specific antibody responses might help to define protective antibody levels as correlate of protective immunity.\nIn conclusion, our in-house flow cytometry–based serological assay has good specificity and sensitivity for the detection of antibodies to SARS-CoV-2. In addition to the nucleotide sequence of the antigen, only readily available reagents were needed to establish the assay. Therefore, the flow cytometric assay may also serve as a blueprint for rapid-response antibody tests against other emerging viral infections."}

{"project":"2_test","denotations":[{"id":"33078221-32284614-28920603","span":{"begin":1507,"end":1509},"obj":"32284614"},{"id":"33078221-32267220-28920604","span":{"begin":1812,"end":1814},"obj":"32267220"},{"id":"33078221-32267220-28920605","span":{"begin":2842,"end":2844},"obj":"32267220"},{"id":"33078221-20943876-28920606","span":{"begin":3239,"end":3241},"obj":"20943876"}],"text":"Discussion\nWhile rigorous measures led to a partial control of the recent SARS-CoV-2 pandemic in some countries, validated serological assays are needed to consolidate those achievements and to support the transition to a post-peak phase. This includes for example diagnostic measures for late/post-infection stages, COVID-19 contact tracing, the assessment of epidemiological aspects, and the evaluation of immunity after infection or in potential vaccine trials. In the recent study, we validated an in-house flow cytometric assay for the detection of SARS-CoV-2S-specific IgM and IgG using sera from PCR-confirmed COVID-19 cases and a collection of control serum samples. In regard to specificity and sensitivity, our flow cytometric assay showed a comparable or even better performance compared to commercial CE-marked serological assays (EuroImmun ELISA and Shenzhen Yhlo CLIA) (Table 1).\nDetection of viral nucleic acids via real-time PCR is the gold standard in the diagnosis of acute SARS-CoV-2 infections. However, despite its reliability early during infection, confirmation of an infection at later time points becomes less reliable. As early as 8 days post-infection, the diagnostic value of serological assays might therefore outperform nucleic acid–based methods [20, 21]. Indeed, also our study showed seroconversion in a longitudinal set of sera from one patient 8 days after the first positive PCR test, although the exact infection date is not clearly defined. As reported before [20, 22, 23], IgM did not generally possess a higher clinical sensitivity compared to IgG, since most of the IgM+ specimen tested in the present study were positive for both isotypes. Detection of SARS-CoV-2-specific IgA was previously reported as more sensitive than detection of IgG in the EuroImmun ELISA kits [14]. However, while this held also true in our study, IgG and IgM measured by the flow cytometric assay were similarly sensitive compared to the IgA ELISA. This higher sensitivity to detect S-specific IgG might be due to the different viral antigens used in the assays. Our flow cytometric assay exploits full-length S protein in its natural conformation and with the respective post-translational modifications due to the expression in mammalian cells. This enables detection of the full spectrum of S-specific antibodies directed against conformational epitopes and glycosylated sites as well, some immunogenic sites possibly missing in truncated, recombinant S1-only proteins as used in the EuroImmun ELISA.\nA potential downside of using full-length S for serological testing might be the detection of cross-reactive antibodies induced by other HCoV. Along this line, some assays detect only antibodies directed against the S1 subunit (like the EuroImmun ELISA) or the receptor-binding domain in order to increase specificity [14, 15]. However, in a collection of sera from individuals that suffered from an infection with an endemic HCoV shortly before blood collection, none was tested positive for SARS-CoV-2 antibodies. In additional 180 specimens sampled before the COVID-19 outbreak, two sera were found to be reactive in the flow cytometric assay. Since endemic HCoV seroprevalence is high in the general population [11] and those two individuals were non-reactive in the commercial S1-specific ELISA, a plausible explanation is cross-reaction of antibodies induced by the endemic HCoVs with the S2 subunit of SARS-CoV-2. Although the reactivity of the two specimens needs to be classified as false-positive detection of SARS-CoV-2 antibodies, these cross-reactive antibodies might possess antiviral activity against COVID-19 and the analysis of cross-protection due to these responses might be an interesting topic for further investigations.\nRegarding the clinical sensitivity, the flow cytometric serology assay detected 100% of IgM- and IgG-positive samples measured by either of the two commercial assays. Only in cases where blood samples were taken at the same day as PCR sampling, all assays (ELISA, CLIA, cytometry) were negative, probably reflecting acute infections prior to development of detectable antibody responses. The lower analytical detection limit of the flow cytometric assay is consistent with its excellent clinical sensitivity.\nSince in the early phase of such a pandemic, there are naturally a limited number of reliable serological test kits available, and there is a high need to expand the portfolio of serology techniques which can be rapidly applied and scaled up. The flow cytometry–based technique to detect SARS-CoV-2 seroconversion presented here fulfills fundamental criteria in regard to sensitivity, specificity, and robustness. The basic requirements needed like cell culture, plastic ware, and a flow cytometer are available in many standard diagnostic and biomedical research labs. Although performing the assay and analyzing the primary data need some trained personal, high-throughput solutions of this method can increase serology testing capacities significantly without competing for ELISA/CLIA kits. Given the large number of antibody assays reaching the market without clearly defined analytical sensitivities, using recombinant ACE-2 Fc protein for standardization is a potential strategy for cross-assay comparisons. Moreover, the quantification of S-specific antibody responses might help to define protective antibody levels as correlate of protective immunity.\nIn conclusion, our in-house flow cytometry–based serological assay has good specificity and sensitivity for the detection of antibodies to SARS-CoV-2. In addition to the nucleotide sequence of the antigen, only readily available reagents were needed to establish the assay. Therefore, the flow cytometric assay may also serve as a blueprint for rapid-response antibody tests against other emerging viral infections."}