Immunoassay for the quantitative determination of antibodies to the SARS-CoV-2 spike protein
Elecsys® Anti-SARS-CoV-2 S is an immunoassay for the in vitro quantitative determination of total antibodies to the SARS-CoV-2 S protein RBD in human serum and plasma. The assay uses a recombinant RBD protein in a double-antigen sandwich assay format, which favors the quantitative determination of high affinity antibodies against SARS-CoV-2. The test is intended as an aid to assess the adaptive humoral immune response, including neutralizing antibodies, to the SARS-CoV-2 S protein after natural infection with SARS-CoV-2 or in vaccine recipients.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), is an enveloped, single-stranded RNA Betacoronavirus. Seven coronaviruses have been identified as agents of human infection, causing disease ranging from mild common cold to severe respiratory failure.1
SARS-CoV-2 is transmitted primarily from person-to-person through respiratory droplets and aerosols.2,3 The incubation period from infection to detectable viral load in the host commonly ranges from two to 14 days.4,5 Detection of viral load can be associated with the onset of clinical signs and symptoms, although a considerable proportion of individuals remain asymptomatic or mildly symptomatic.6-8 The interval during which an individual with COVID-19 is infectious has not yet been clearly established, however, transmission from symptomatic, asymptomatic, and pre-symptomatic individuals has been well described.9-11
Coronavirus genomes encode 4 main structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N). The S protein is a very large transmembrane protein that assembles into trimers to form the distinctive surface spikes of coronaviruses. Each S monomer consists of an N-terminal S1 subunit and a membrane-proximal S2 subunit. The virus gains entry to the host cell through binding of the S protein to the angiotensin-converting enzyme 2 (ACE2) receptor, which is present on the surface of numerous cell types including the alveolar type II cells of the lung and epithelial cells of the oral mucosa.12,13 Mechanistically, ACE2 is engaged by the receptor-binding domain (RBD) on the S1 subunit.14,15
Upon infection with SARS-CoV-2, the host usually mounts an immune response against the virus, typically including production of specific antibodies against viral antigens. IgM and IgG antibodies against SARS-CoV-2 appear to arise nearly simultaneously in blood.16 There is significant inter-individual difference in the levels and chronological appearance of antibodies in COVID-19 patients, but median seroconversion has been observed at approximately two weeks.17-20
After infection or vaccination, the binding strength of antibodies to antigens increases over time - a process called affinity maturation21. High‑affinity antibodies can elicit neutralization by recognizing and binding specific viral epitopes22,23. Antibodies against SARS‑CoV‑2 with strong neutralizing capacity, especially potent if directed against the RBD, have been identified.24-27 Numerous vaccines for COVID-19 are in development, many of which focus on eliciting an immune response to the RBD.28-30
A total of 1,610 samples from 402 symptomatic patients (including 297 samples from 243 hospitalized patients) with a PCR confirmed SARS-CoV-2 infection were tested with the Elecsys® Anti-SARS-CoV-2 S assay. One or more sequential samples from these patients were collected at various time points after PCR confirmation.
1,423 of the tested samples had a sampling date of 14 days or later after diagnosis with PCR. 1,406 of these 1,423 samples were determined with ≥0.8 U/mL in the Elecsys® Anti‑SARS‑CoV‑2 S assay and hence considered positive, resulting in a sensitivity of 98.8 % (95 % CI: 98.1 – 99.3 %) in this sample cohort.
A total of 1,610 samples from 402 symptomatic patients (including 297 samples from 243 hospitalized patients) with a PCR confirmed SARS-CoV-2 infection were tested with the Elecsys® Anti-SARS-CoV-2 S assay. One or more sequential samples from these patients were collected at various time points after PCR confirmation.
1,423 of the tested samples had a sampling date of 14 days or later after diagnosis with PCR. 1,406 of these 1,423 samples were determined with ≥0.8 U/mL in the Elecsys® Anti‑SARS‑CoV‑2 S assay and hence considered positive, resulting in a sensitivity of 98.8 % (95 % CI: 98.1 – 99.3 %) in this sample cohort.
Days post PCR confirmation
| Days post PCR confirmation | N | Non-reactive | Sensitivity (95 % CI*) |
| 0-6 days | 35 | 4 | 88.6 % (73.3 – 96.8 %) |
| 7-13 days | 152 | 22 | 85.5 % (78.9 – 90.7 %) |
| 14-20 days | 130 | 14 | 89.2 % (82.6 – 94.0 %) |
| 21-27 days | 176 | 3 | 98.3 % (95.1 – 99.7 %) |
| 28-34 days | 197 | 0 | 100 % (98.1 – 100.0 %) |
| ≥35 days | 920 | 0 | 100 % (99.6 – 100.0 %) |
A total of 1,100 potentially cross-reactive samples collected before October 2019, including anti-MERS-CoV positive samples, samples from individuals with common cold symptoms, and samples from individuals confirmed to be infected with one of the four common cold coronaviruses were tested with the Elecsys® Anti-SARS-CoV-2 S assay. Overall specificity in this cohort of potentially cross-reactive samples was 100 % (95 % CI: 99.7 – 100.0 %).
A total of 1,100 potentially cross-reactive samples collected before October 2019, including anti-MERS-CoV positive samples, samples from individuals with common cold symptoms, and samples from individuals confirmed to be infected with one of the four common cold coronaviruses were tested with the Elecsys® Anti-SARS-CoV-2 S assay. Overall specificity in this cohort of potentially cross-reactive samples was 100 % (95 % CI: 99.7 – 100.0 %).
| Cohort | N | Reactive | Specificity (95% CI) |
| MERS-CoV* | 7 | 0 | 100 % (59.0 – 100.0 %) |
| Common cold panel** | 21 | 0 | 100 % (83.4 – 100.0 %) |
| Coronavirus panel*** | 94 | 0 | 100 % (96.2 – 100.0 %) |
| Other potentially cross-reactive samples**** | 978 | 0 | 100 % (99.6 – 100.0 %) |
| Overall | 1,100 | 0 | 100 % (99.7 – 100.0 %) |
A total of 5,991 samples from diagnostic routine and blood donors drawn before October 2019 were tested with the Elecsys® Anti-SARSCoV-2 S assay. Overall specificity in this cohort of pre-pandemic samples was 99.98 % (95 % CI: 99.91 – 100 %).
A total of 5,991 samples from diagnostic routine and blood donors drawn before October 2019 were tested with the Elecsys® Anti-SARSCoV-2 S assay. Overall specificity in this cohort of pre-pandemic samples was 99.98 % (95 % CI: 99.91 – 100 %).
| Cohort | N | Reactive | Specificity (95 % CI) |
| Diagnostic routine | 2,528 | 0 | 100 % (99.85 – 100.0 %) |
| US blood donors | 2,713 | 1 | 99.96 % (99.79 – 100.0 %) |
| African blood donors | 750 | 0 | 100 % (99.51 – 100.0 %) |
| Overall | 5,991 | 1 | 99.98 % (99.91 – 100.0 %) |
After vaccination with the Moderna vaccine Spikevax® (mRNA‑1273) and the Pfizer‑BioNTech vaccine Comirnaty® (BNT162b2), applying the respectively approved 2-dose vaccination scheme, the antibody response in vaccinated, infection-naïve individuals was assessed using the Elecsys® Anti-SARS-CoV-2 S assay at three time-points: pre-vaccination (baseline), 21 days post 1st vaccination dose, and 14 days post 2nd vaccination dose. Following vaccination, rapidly rising antibody titers, indicating a strong humoral immune response to vaccination, were observed. All individuals that had been seronegative at baseline seroconverted after vaccination.
After vaccination with the Moderna vaccine Spikevax® (mRNA‑1273) and the Pfizer‑BioNTech vaccine Comirnaty® (BNT162b2), applying the respectively approved 2-dose vaccination scheme, the antibody response in vaccinated, infection-naïve individuals was assessed using the Elecsys® Anti-SARS-CoV-2 S assay at three time-points: pre-vaccination (baseline), 21 days post 1st vaccination dose, and 14 days post 2nd vaccination dose. Following vaccination, rapidly rising antibody titers, indicating a strong humoral immune response to vaccination, were observed. All individuals that had been seronegative at baseline seroconverted after vaccination.
In a study investigating COVID‑19 convalescent plasma for virus neutralization capacity, plasma donations from convalescent donors after a SARS‑CoV‑2 infection were analyzed for whole virus neutralizing potential using an in vitro plaque reducing neutralization (PRNT) assay (BROAD Institute, USA). Presence of 50 % neutralization (NT50) at a sample dilution of >1:20 identified functional virus neutralization in vitro. 390 donations, including cross-sectional and longitudinal sample panels, were analyzed by PRNT and compared to Elecsys® Anti‑SARS‑CoV‑2 S assay results by applying two different thresholds: one representing the assay’s cutoff for detecting presence of RBD-specific antibodies (0.8 U/mL), and one based on optimized correlation with detection of virus neutralizing effects (15 U/mL).
In a study investigating COVID‑19 convalescent plasma for virus neutralization capacity, plasma donations from convalescent donors after a SARS‑CoV‑2 infection were analyzed for whole virus neutralizing potential using an in vitro plaque reducing neutralization (PRNT) assay (BROAD Institute, USA). Presence of 50 % neutralization (NT50) at a sample dilution of >1:20 identified functional virus neutralization in vitro. 390 donations, including cross-sectional and longitudinal sample panels, were analyzed by PRNT and compared to Elecsys® Anti‑SARS‑CoV‑2 S assay results by applying two different thresholds: one representing the assay’s cutoff for detecting presence of RBD-specific antibodies (0.8 U/mL), and one based on optimized correlation with detection of virus neutralizing effects (15 U/mL).
| Virus Neutralization Test (PRNT) | ||||
| Neutralizing (NT50 ≥ 1:20) |
Non-neutralizing | Total | ||
| Elecsys® Anti‑SARS‑CoV‑2 S | ≥0.8 U/mL | 356 | 4 | 360 |
| <0.8 U/mL | 2 | 28 | 30 | |
| Total | 358 | 32 | 390 | |
| Percent Positive Agreement | 99.4 % (98.0 – 99.9 %) | |||
| Percent Negative Agreement | 87.5 % (71.0 – 96.5 %) | |||
| Positive Predictive Value | 98.9 % (97.2 – 99.7 %) | |||
| Elecsys® Anti‑SARS‑CoV‑2 S | ≥15 U/mL | 331 | 0 | 331 |
| <15 U/mL | 27 | 32 | 59 | |
| Total | 358 | 32 | 390 | |
| Percent Positive Agreement | 92.5 % (89.2 – 95.0 %) | |||
| Percent Negative Agreement |
100 % (89.1 – 100.0 %) | |||
| Positive Predictive Value | 100 % (98.9 – 100.0 %) | |||
References
