Background Quantitative serological assays detecting response to SARS-CoV-2 infection are urgently needed to quantify immunity. This study analyzed the performance and correlation of two independent quantitative anti-S1 assays in oligo-/asymptomatic individuals from a previously characterized population-based cohort. Methods A total of 362 samples included 108 from individuals who had viral RNA detected in pharyngeal swabs, 111 negative controls and 143 samples with positive serology but not confirmed by RT-PCR. Blood plasma was tested with quantitative assays Euroimmun Anti-SARS-CoV-2 QuantiVac ELISA (IgG) (EI-S1-IgG-quant) and Roche Elecsys ® Anti-SARS-CoV-2 CoV-2 S (Ro-RBD-Ig-quant), which were compared with each other and with confirmatory tests, including wild-type virus micro-neutralization (NT) and GenScript ® cPass™. Results were analyzed using square roots R of coefficients of determination for association among continuous variables and non-parametric tests for paired comparisons. Results Quantitative anti-S1 serology correlated well with each other (96%/97% for true-positives and true-negatives, respectively). Antibody titers decreased over time (from <30 days to >240 days after initial positive RT-PCR). Agreement with GenScript-cPass was 96%/99% for true-positives and true-negatives, respectively, for Ro-RBD-Ig-quant and 93%/97% for EI-S1-IgG-quant. Ro-RBD-Ig-quant allowed a distinct separation between positive and negative values, and less non-specific reactivity compared with EI-S1-IgG-quant. Raw values (with 95% CI) ≥28.7 U/mL (22.6–36.4) for Ro-RBD-Ig-quant and ≥49.8 U/mL (43.4–57.1) for EI-S1-IgG-quant predicted virus neutralization >1:5 in 95% of cases. Conclusions Both quantitative anti-S1 assays, Ro-RBD-Ig-quant and EI-S1-IgG-quant, may replace direct neutralization assays in quantitative measurement of immune protection against SARS-CoV-2 in certain circumstances in the future. Key points Two quantitative anti-S1 assays showed similar performance and a high level of agreement with direct virus neutralization and surrogate neutralization tests, arguing for their utility in quantifying immune protection against SARS-CoV-2.
Abstract Background The Elecsys ® Anti-SARS-CoV-2 immunoassay (Roche Diagnostics) was developed to provide an accurate and reliable method for the detection of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated the sensitivity, specificity, and cross-reactivity of the Elecsys Anti-SARS-CoV-2 immunoassay. Methods The performance of the Elecsys Anti-SARS-CoV-2 immunoassay was assessed at Roche Diagnostics (Penzberg, Germany). Sensitivity was evaluated using anonymised residual frozen samples from patients who had previously tested positive for SARS-CoV-2 infection by polymerase chain reaction (PCR); one or more consecutive samples were collected from patients at various timepoints after PCR confirmation. Specificity was evaluated using anonymised unselected residual frozen samples from routine diagnostic testing or from blood donors; all samples were collected before December 2019 and thus deemed negative for SARS-CoV-2-specific antibodies. Cross-reactivity was evaluated using anonymised frozen samples containing a wide range of potentially cross-reacting analytes, which were purchased from commercial vendors. For sensitivity and specificity, point estimates and 95% confidence intervals (CIs) were calculated. Results Sensitivity of the Elecsys Anti-SARS-CoV-2 immunoassay in 496 samples from 102 patients with prior PCR-confirmed SARS-CoV-2 infection was 99.5% (95% CI 97.0–100.0) at ≥14 days after PCR confirmation. Overall specificity in 10,453 samples from routine diagnostic testing (n = 6305) and blood donors (n = 4148) was 99.80% (95% CI 99.69–99.88). Only 4/752 samples containing potential cross-reacting analytes were reactive with the Elecsys Anti-SARS-CoV-2 immunoassay, resulting in an overall specificity in this cohort of 99.5% (95% CI 98.6–99.9). Conclusion The Elecsys Anti-SARS-CoV-2 immunoassay demonstrated a sensitivity of 99.5% at ≥14 days after PCR confirmation and a very high specificity of 99.80%. Our findings support the use of the Elecsys Anti-SARS-CoV-2 immunoassay as a tool for the identification of past SARS-CoV-2 infection, including in populations with a low disease prevalence. Required information for submission system Ethical guidelines The study was conducted in accordance with applicable regulations, including relevant European Union directives and regulations, and the principles of the Declaration of Helsinki. All samples, excluding the specimens that were provided by commercial sample vendors, were transferred to Roche following anonymisation. For studies with anonymised leftover specimens, no ethics committee vote is required. A statement was obtained from the Ethics Committee of the Landesä rztekammer Bayern confirming that there are no objections against the transfer and the coherent use of the anonymised leftover samples. Research reporting guidelines Please see separate STARD checklist Data availability statement Qualified researchers may request access to individual patient level data through the clinical study data request platform ( https://vivli.org/ ). Further details on Roche’s criteria for eligible studies are available here: https://vivli.org/members/ourmembers/ . For further details on Roche’s Global Policy on the Sharing of Clinical Information and how to request access to related clinical study documents, see here: https://www.roche.com/research_and_development/who_we_are_how_we_work/clinical_trials/our_commitment_to_data_sharing.htm .
The use of antibody titers against SARS-CoV-2, as a method of estimating subsequent infection following infection or vaccination, is unclear. Here, we investigate whether specific levels of antibodies, as markers of adaptive immunity, can serve to estimate the risk of symptomatic SARS-CoV-2 (re-) infection. In this real-world study, laboratory data from individuals tested for SARS-CoV-2 antibodies under routine clinical conditions were linked through tokenization to a United States medical insurance claims database to determine the risk of symptomatic/severe SARS-CoV-2 infection outcomes. Antibody titer levels were determined using the Elecsys® Anti-SARS-CoV-2 S assay. Study outcomes included the first symptomatic SARS-CoV-2 infection (per ICD-10 diagnostic codes, occurring ≥ 7 days post-antibody titer test), and severe SARS-CoV-2 infection, characterized by adverse outcomes including hospitalization, intensive care unit admission, intubation, mechanical ventilation, or death within 30 days of infection. All outcomes were assessed for 12 months following antibody measurement. Hazard ratios of subsequent symptomatic and severe infections were estimated using Cox regression with inverse probability weighting. Of 268,844 individuals with antibody data (April 2021–June 2022), those with levels ≥ 0.8 to < 1,000 U/mL had a 42% reduced risk of symptomatic infection within 12 months, compared with < 0.8 U/mL (HR = 0.58, 95% CI [0.55, 0.61]). The risk decreased by 53% (HR = 0.47, 95% CI [0.45, 0.49]) with ≥ 1000 to < 2500 U/mL and by 62% (HR = 0.38 [0.36, 0.39]) for ≥ 2500 U/mL. Risk of severe SARS-CoV-2 outcomes was also reduced. Subgroup analyses showed a consistent association between antibody levels and infection risk, by immune status and age. Clinically meaningful thresholds of antibody titers varied between Delta and Omicron infections. Higher antibody titer levels indicated reduced risk of developing symptomatic or severe COVID-19. Titers of ≥ 2500 U/mL indicated a 62–87% reduced infection risk. The quantitative determination of antibody titers allowed scaling of the correlate of risk to new variants.
The ability to quantify an immune response after vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential. This study assessed the clinical utility of the quantitative Roche Elecsys
Abstract Background There is a need for automated, high throughput assays to quantify immune response after vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study assessed the combined utility of the Roche assays, Elecsys ® Anti-SARS-CoV-2 S (ACOV2S) and Elecsys Anti-SARS-CoV-2 (ACOV2N) using samples from the 2019-nCoV vaccine (mRNA-1273, Spikevax™) phase 2 trial ( NCT04405076 ). Methods Samples from 593 healthy participants in two age cohorts (18–54 years and ≥55 years), who received two injections with either placebo (n=198) or mRNA-1273 at a dose of either 50 μg (n=197) or 100 μg (n=198), were collected at Days 1 (first vaccination), 15, 29 (second vaccination), 43 and 57. ACOV2S results were used to assess the humoral response to vaccination in different clinical trial subgroups and were compared to a live virus microneutralization assay. Sample panels from patients with evidence of previous or concomitant infection (as identified using ACOV2N) or with an inconsistent antibody response pattern were analyzed separately. Results Receptor-binding domain (RBD)-specific antibodies were readily detectable by ACOV2S for the vast majority of participants (174/189 [50 μg dose group] and 178/192 [100 μg]) at the first time point of assessment, with non-converters predominantly older in age. Complete seroconversion for all participants was observed at the subsequent timepoint (Day 29) and before administration of the second dose of vaccine. Two weeks after the first vaccine dose (Day 15), geometric mean concentration (GMC) of antibody levels were 1.37-fold higher in the 100 μg compared with the 50 μg dose group; this difference reduced to 1.09-fold two weeks after the second dose (Day 43). In both the 50 μg and 100 μg dose groups, a more pronounced response was observed in the younger versus the older age group on Day 15 (2.49-fold and 3.94-fold higher GMC, respectively) and Day 43 (1.35-fold and 1.50-fold higher GMC). Few subjects had a previous or concomitant natural SARS-CoV-2-infection (n=8). Vaccination of pre-infected individuals boosted the immune response to very high ACOV2S results compared to infection-naïve vaccine recipients. ACOV2S measurements were strongly correlated with those from the live microneutralization assay (Pearson’s r=0.779; p<0.0001) and good qualitative agreement was achieved (100% positive and 91.8% negative percentage agreement; 90.0% positive and 100% negative predictive value). Conclusion The results from this study confirmed that ACOV2S is a highly valuable assay for the tracking of vaccine-related immune responses. Combined application with ACOV2N enables serologic monitoring for breakthrough infection or stratification of previous natively-infected individuals. The adaptive measuring range and high resolution of ACOV2S allows for the early identification of seroconversion as well as for resolution of very high titers and detection of longitudinal differences between age and dose groups. Additionally, good correlation of ACOV2S with live virus microneutralization indicates the utility of ACOV2S as a reliable estimate of neutralization capacity in routine diagnostic settings.
Background Automated, high throughput assays are required to quantify the immune response after infection with or vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study on the Roche Elecsys ® Anti-SARS-CoV-2 S (ACOV2S) assay provides insights on the assay design and performance. Methods The ACOV2S assay quantifies antibodies to the receptor-binding domain of the SARS-CoV-2 spike protein. The assigned units and the underlying standardization were compared to the international reference standard in BAU/mL. Assay specificity was assessed in samples (n=5981) collected prior to the COVID-19 pandemic and in samples from patients with non-COVID-19 respiratory infections (n=697) or other infectious diseases (n=771). Sensitivity was measured in 1313 samples from patients with mild COVID-19 and 297 samples from patients hospitalized with COVID-19. Comparison of results was performed to a comparator semi-quantitative anti-S1 assay of indirect detection format as well as a commercially available and an in-house version of a surrogate neutralization assay (ACE2-RBD). Results The originally assigned units for the ACOV2S assay were shown to be congruent to the units of the First International WHO Standard for anti-SARS-CoV-2 immunoglobulins. Overall specificity was 99.98% with no geographical differences noted and no loss of specificity in samples containing potentially cross-reacting antibodies. High sensitivity was observed, with 98.8% of samples reported to be reactive >14 days after infection and sustained detection of antibodies over time. For all samples, ACOV2S titers and neutralization capacities developed with comparable dynamics. Robust standardization and assay setup enable excellent reproducibility of results, independent of lot or analyzer used. Conclusion The results from this study confirmed that ACOV2S is a highly sensitive and specific assay and correlates well with surrogate neutralization assays. The units established for ACOV2S are also interchangeable with the units of the First International WHO Standard for anti-SARS-CoV-2 immunoglobulins. Worldwide availability of the assay and analyzers render ACOV2S a highly practical tool for population-wide assessment and monitoring of the humoral response to SARS-CoV-2 infection or vaccination.
COVID-19 has highlighted challenges in the measurement quality and comparability of serological binding and neutralization assays. Due to many different assay formats and reagents, these measurements are known to be highly variable with large uncertainties. The development of the WHO international standard (WHO IS) and other pool standards have facilitated assay comparability through normalization to a common material but does not provide assay harmonization nor uncertainty quantification. In this paper, we present the results from an interlaboratory study that led to the development of (1) a novel hierarchy of data analyses based on the thermodynamics of antibody binding and (2) a modeling framework that quantifies the probability of neutralization potential for a given binding measurement. Importantly, we introduced a precise, mathematical definition of harmonization that separates the sources of quantitative uncertainties, some of which can be corrected to enable, for the first time, assay comparability. Both the theory and experimental data confirmed that mAbs and WHO IS performed identically as a primary standard for establishing traceability and bridging across different assay platforms. The metrological anchoring of complex serological binding and neuralization assays and fast turn-around production of an mAb reference control can enable the unprecedented comparability and traceability of serological binding assay results for new variants of SARS-CoV-2 and immune responses to other viruses.
Abstract Background Automated, high throughput assays are required to quantify the immune response after infection with or vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study on the Roche Elecsys ® Anti-SARS-CoV-2 S (ACOV2S) assay provides insights on the assay design and performance. Methods The ACOV2S assay quantifies antibodies to the receptor-binding domain of the SARS-CoV-2 spike protein. The assigned units and the underlying standardization were compared to the international reference standard in BAU/mL. Assay specificity was assessed in samples (n=5981) collected prior to the COVID-19 pandemic and in samples from patients with non-COVID-19 respiratory infections (n=697) or other infectious diseases (n=771). Sensitivity was measured in 1313 samples from patients with mild COVID-19 and 297 samples from patients hospitalized with COVID-19. Comparison of results was performed to a comparator semi-quantitative anti-S1 assay of indirect detection format as well as a commercially available and an in-house version of a surrogate neutralization assay (ACE2-RBD). Results The originally assigned units for the ACOV2S assay were shown to be congruent to the units of the First International WHO Standard for anti-SARS-CoV-2 immunoglobulins. Overall specificity was 99.98% with no geographical differences noted and no loss of specificity in samples containing potentially cross-reacting antibodies. High sensitivity was observed, with 98.8% of samples reported to be reactive >14 days after infection and sustained detection of antibodies over time. For all samples, ACOV2S titers and neutralization capacities developed with comparable dynamics. Robust standardization and assay setup enable excellent reproducibility of results, independent of lot or analyzer used. Conclusion The results from this study confirmed that ACOV2S is a highly sensitive and specific assay and correlates well with surrogate neutralization assays. The units established for ACOV2S are also interchangeable with the units of the First International WHO Standard for anti-SARS-CoV-2 immunoglobulins. Worldwide availability of the assay and analyzers render ACOV2S a highly practical tool for population-wide assessment and monitoring of the humoral response to SARS-CoV-2 infection or vaccination.
Quantitative serological assays detecting response to SARS-CoV-2 are needed to quantify immunity. This study analyzed the performance and correlation of two quantitative anti-S1 assays in oligo-/asymptomatic individuals from a population-based cohort.In total, 362 plasma samples (108 with reverse transcription-polymerase chain reaction [RT-PCR]-positive pharyngeal swabs, 111 negative controls, and 143 with positive serology without confirmation by RT-PCR) were tested with quantitative assays (Euroimmun Anti-SARS-CoV-2 QuantiVac enzyme-linked immunosorbent assay [EI-S1-IgG-quant]) and Roche Elecsys® Anti-SARS-CoV-2 S [Ro-RBD-Ig-quant]), which were compared with each other and confirmatory tests, including wild-type virus micro-neutralization (NT) and GenScript®cPass™. Square roots R of coefficients of determination were calculated for continuous variables and non-parametric tests were used for paired comparisons.Quantitative anti-S1 serology correlated well with each other (true positives, 96%; true negatives, 97%). Antibody titers decreased over time (< 30 to > 240 days after initial positive RT-PCR). Agreement with GenScript-cPass was 96%/99% for true positives and true negatives, respectively, for Ro-RBD-Ig-quant and 93%/97% for EI-S1-IgG-quant. Ro-RBD-Ig-quant allowed distinct separation between positives and negatives, and less non-specific reactivity versus EI-S1-IgG-quant. Raw values (95% CI) ≥ 28.7 U/mL (22.6-36.4) for Ro-RBD-Ig-quant and ≥ 49.8 U/mL (43.4-57.1) for EI-S1-IgG-quant predicted NT > 1:5 in 95% of cases.Our findings suggest both quantitative anti-S1 assays (EI-S1-IgG-quant and Ro-RBD-Ig-quant) may replace direct neutralization assays in quantitative measurement of immune protection against SARS-CoV-2 in certain circumstances. However, although the mean antibody titers for both assays tended to decrease over time, a higher proportion of Ro-RBD-Ig-quant values remained positive after 240 days.
