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Comparison of two automated immunoassays for the detection of SARS-CoV-2 nucleocapsid antibodies

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel member of the coronavirus family that caused the global coronavirus 2019 (COVID-19) pandemic. The prevalence remains largely unknown because of early testing supply shortages. Although it cannot currently be used to determine level of immunity, antibody testing can contribute to epidemiological studies, identify convalescent plasma donors, or satisfy curiosity about previous exposure to the virus. Methods 407 samples collected from hospitalized inpatients with and without a confirmed SARS-CoV-2 infection, 170 remnant clinical specimens collected and frozen prior to the COVID-19 outbreak, and paired serum and plasma samples from 23 convalescent plasma donors were used to determine performance characteristics of the Abbott SARS-CoV-2 IgG and Roche Elecsys Anti-SARS-CoV-2 assays. The sensitivity, specificity, imprecision, interferences, and sample stability were determined. These assays were then used to characterize the antibody response in serial samples from 20 SARS-CoV-2 positive inpatients. Results Both assays exhibited 100% specificity (95% CI; 99.05 – 100.00), giving no positive results in 170 specimens collected before July 2019 and 215 specimens from patients without a confirmed SARS-CoV-2 infection. Differences between platforms were most notable in SARS-CoV-2 positive samples. Roche offered higher sensitivity in convalescent plasma donors at 95.7% (95% CI; 78.1 – 99.9) versus 91.3% (95% CI; 72.0 – 98.9) but Abbott detected antibodies in two immunocompromised patients whereas Roche did not. The Roche and Abbott platforms also exhibited different trends in antibody signal for a subset of patients. Conclusions Both the Abbott and Roche platforms offer excellent specificity but different trends in antibody signal may reflect qualitative differences in the types of antibodies recognized by the two assays. Negative serologic results do not exclude previous SARS-CoV-2 infection.


Introduction
A respiratory pneumonia caused by a novel human coronavirus was discovered in Wuhan, China in December 2019. Shortly thereafter, the genetic sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was released and confirmed to be a highly pathogenic virus causing the coronavirus disease 2019 (COVID- 19) outbreak (1). The early outbreak exhibited exponential growth as it spread worldwide with a basic reproductive number (R0) estimated to be between 1.4 -6.5 (2)(3)(4)(5)(6)(7). The first case reported in the United States was on January 19, 2020 (8) and the virus spread to all 50 states by mid-March. Given the degree of global spread, the World Health Organization declared a pandemic on March 11, 2020 (9).
The identifying clinical features of SARS-CoV-2 infection include fever and respiratory symptoms, accompanied by radiographic pulmonary features appearing three to seven days after exposure. Symptoms range from mild to critical and some present with only nonrespiratory symptoms, such as mild fatigue, headache, or diarrhea, while others present with life threatening symptoms such as septic shock, multiple organ failure, or fatal pneumonia (10,11). Preliminary data suggest that after approximately two weeks of illness, there may be a positive correlation between clinical severity and antibody signal (12,13).
Active infection with SARS-CoV2 is diagnosed with nucleic acid amplification testing of the virus collected with either throat or nasopharayngeal swabs (14). Rapid development of molecular testing was critical in identifying affected individuals so they could self-quarantine and mitigate the spread of the disease. Due to shortages of collection and reagent supplies, this testing was not widely available at the start of the outbreak. As a result of these shortages, the total number of infected individuals remains unknown. However, it is possible to estimate prevalence of this virus with serology testing.
SARS-CoV-2 is an enveloped positive sense single-stranded RNA virus that, under microscopy, resembles the shape of a crown due to its spike structural proteins (10). The coronavirus particle also contains a nucleoprotein (N) that encapsulates the RNA, a small envelope protein that surrounds the helical nucleocapsid, and a membrane glycoprotein that is embedded in the envelope (15). The S protein is an appealing target for antibody assay design because it is immunogenic and is the target of neutralizing antibodies. Despite this, many assays, including the Abbott IgG and Roche total antibody assays, target the N protein. This is likely due to the increased sensitivity (16) and specificity offered by the lack of sequence conservation throughout the N protein among coronaviruses (17).
To date, there are numerous SARS-CoV-2 serology assays available on the market. However, only a subset have received FDA Emergency Use Authorization (EUA), of which only a few are available on high throughput, automated platforms that can meet the demands of a large clinical laboratory. Similar to other recent publications (18)(19)(20), we conducted a head-to-head comparison of the Abbott SARS-CoV-2 IgG and Roche Elecsys Anti-SARS-CoV-2 assays. We compared the performance characteristics and used the validated testing platforms to characterize the antibody response of convalescent plasma donors and the seroconversion of inpatients with known SARS-CoV-2 infection.

Hospitalized Inpatients
Four hundred seven remnant serum and lithium heparin plasma specimens collected in gel separator tubes for clinical purposes from hospitalized patients with and without confirmed SARS-CoV-2 infection were removed from refrigerated storage within seven days of collection.
All patients included in this study were tested for SARS-CoV-2 infection with one of three molecular diagnostic methods: the CDC format laboratory developed test, the Abbott RealTime SARS-CoV-2 m2000 assay, or the Diasorin Simplexa COVID-19 assay. Two hundred fifteen of these specimens were collected from 155 unique patients with a documented negative SARS-CoV-2 molecular diagnostic result generated on the same day or one day prior to serum or plasma specimen collection. Both serum and plasma were available for 60 patients while only plasma was available for the remaining 94 patients. The remaining 193 remnant specimens were collected from 20 unique, symptomatic patients with a previously documented positive SARS-CoV-2 molecular diagnostic result. Serum and plasma samples were aliquoted into sealed plastic tubes and frozen at -80°C until further use (no longer than 3 weeks in frozen storage).

Pre-SARS-CoV-2 Outbreak
170 remnant serum and lithium heparin plasma specimens collected in gel separator tubes for clinical purposes between September 2017 and June 2019 had been sequestered in -20°C storage for future validation projects. Consistent with internal laboratory practice, the specimens had been removed from refrigerated storage within seven days of collection, transferred to sealed plastic tubes, and stored at -20°C until further use.

Convalescent Plasma Donors
Paired serum and lithium heparin plasma specimens collected in gel separator tubes were collected at least 14 days after resolution of symptoms from 23 unique individuals with a documented positive SARS-CoV-2 molecular diagnostic result. Serum and plasma samples were aliquoted into sealed plastic tubes and frozen at -80°C until further use (no longer than 3 weeks in frozen storage).

Stability Study
One serum and one lithium heparin specimen from each of three convalescent plasma donors were kept in the original gel separator tubes and stored at 4°C. The specimens were subjected to SARS-CoV-2 serologic testing on days 0-4, 6-7 and 14 after collection.

Institutional Review
This study was approved by the Dartmouth-Hitchcock Institutional Review Board.

Specimen Handling and Data Collection
With the exception of refrigerated specimens used for the stability study, specimens described above were thawed at ambient temperature and briefly centrifuged to remove precipitated material. Specimens were tested using the Abbott SARS-CoV-2 IgG assay (06R8620, Abbott Laboratories Diagnostics Division, Abbott Park, IL) on an Architect i1000 instrument and the Roche Elecsys Anti-SARS-CoV-2 assay (09203079190, Roche Diagnostics, Indianapolis, IN) on a cobas e801 instrument. Individual specimens were tested on both analytical platforms on the same day, and held at refrigerated temperature if they were not tested on the day they were removed from the freezer. All samples were tested within five days after removal from frozen storage.

Roche Elecsys Anti-SARS-CoV-2 Method
The Roche anti-SARS-CoV-2 method uses a bridge format in which patient antibody links biotinylated SARS-CoV-2-specific antigen and ruthenium-labeled SARS-CoV-2-specific antigen to form an antigen -antibody -antigen sandwich complex.

Abbott SARS-CoV-2 IgG Method
The Abbott SARS-CoV-2 IgG method is a two-step immunoassay in which SARS-CoV-2 antigencoated paramagnetic microparticles are exposed to patient sample and bound by anti-SARS-CoV-2 antibodies. (22) Following a wash step, acridinium-labeled anti-human IgG is added and allowed to bind to IgG that remains bound to the washed microparticles.

Negative and Positive Patient Pools
Negative and positive pools were made by combining Li-heparin plasma samples from individuals with documented "Not Detected" or "Detected" SARS-CoV-2 molecular diagnostic test results. SARS-CoV-2 antibody testing was performed on the Abbott Architect to determine the absence or presence of antibody. Pools were aliquoted into sealed plastic vials and stored at -20°C until use.

Comparison of Serum vs. Li-Heparin plasma and Interfering Substances
To verify that serum and Li-Heparin plasma collected in gel-separator tubes would produce comparable results we performed a direct comparison of approximately 100 pairs of matched serum and plasma samples collected from convalescent plasma donors and inpatients as described above.

Inpatient seroconversion
The antibody signals from serial plasma samples collected from 20 inpatients with confirmed SARS-CoV-2 infection were measured on both the Abbott and Roche platforms ( Figure 1

Abbott and Roche performance characteristics
The performance characteristics of both the Abbott and Roche assays were evaluated ( Table 2).
We investigated the effects of sample hemolysis, icterus, and lipemia on both assays. Our studies did not demonstrate any interference (less than 10% bias) up to an H-index of 660, Iindex of 48, or L-index of 1800, the highest concentrations tested for each interferent. To evaluate specimen stability, we tested three pairs of plasma and serum samples (n = 6 total) at concentrations spanning the dynamic range. When stored at 4⁰C, these samples were stable for  Table 3. The Abbott assay exhibited an imprecision ranging from 1.5-2.3% CV with the exception of the Abbott negative QC (7.9% CV). This is likely due to the low S/C of the negative Abbott QC, which ranged from just 0.05 -0.07 with an SD of 0.004. The Roche assay exhibited a consistent imprecision ranging from 3.3-3.7% CV for all material tested. Imprecision was determined over two separate lots and at least two calibrations on the Roche platform but only one lot and one calibration on the Abbott platform.
Matched serum and plasma samples were collected and compared to validate both as acceptable sample types. Consistent results were observed between matrices indicating that both were appropriate for clinical use. Of the approximately 100 matched serum and plasma samples tested, only a single pair of samples generated discrepant qualitative results on the Abbott platform, falling slightly above the threshold for positivity in plasma (S/C 2.11) but just below (S/C 1.29) in serum. Otherwise, the use of serum or plasma did not change the reported result on either platform. No statistically significant difference (two-tailed paired t-test) existed between serum and plasma samples.

Discussion
As it is yet unknown whether anti-SARS-CoV-2 antibodies confer immunity, nor is a vaccine available, serologic testing is currently limited to one of three applications: evaluating prospective convalescent plasma donors, conducting large-scale epidemiologic studies and satisfying the curiosity of individuals who experienced upper respiratory symptoms but were unable to obtain molecular diagnostic testing while symptomatic. According to the manufacturers' package inserts, both the Roche and Abbott assays are suitable for use in these applications as they demonstrated excellent and nearly identical diagnostic performance characteristics despite very different assay designs. In this study, using a single cohort of patient samples tested on both analytical platforms, we confirmed the manufacturers' findings of >99% specificity but observed somewhat lower sensitivity. In our study consisting of 385 SARS-CoV-2 PCR negative samples, we did not find any false positive test results for either assay. While this does confirm the manufacturers' stated specificity of >99%, we cannot claim a true specificity of 100% as it is understood that this likely represents a sampling artifact, and if a larger cohort were tested we would likely encounter some false positive results. Furthermore, we observed important differences in assay performance that may help guide assay selection at a future date when serologic testing is used to monitor immune response in infected patients.
Two of 20 SARS-CoV-2-positive inpatients were immunocompromised (patient 11: chemotherapy, and patient 12: anti-CD20 monoclonal therapy) and both generated consistently positive results on the Abbott platform and negative results on the Roche platform throughout their admissions. While the exact reason for this discrepancy is unclear, it may have important implications for the selection of test methods to evaluate the serologic response in other immunocompromised patients. We did not attempt to assess the affinity of the antibodies produced in these patients and we acknowledge that one limitation of our study is the small number of immunocompromised patients. Further evaluation of these assays in this patient population will be required to definitively establish the cause of the discrepant results.
Different trends in antibody response were also observed between the two assays in the SARS-CoV-2-positive inpatient group. All patients exhibited either a continually increasing or unchanging antibody signal on the Abbott platform over the course of their admission. While 16 patients exhibited one of these two patterns on the Roche platform, four patients (Patients 7-10) showed an initial rise in antibody signal, followed by a gradual decrease to a steady state plateau. This may reflect disappearance of the IgM contribution to the Roche total antibody signal or may indicate a transition in the quality or number of IgG antibodies being produced, resulting in decreasing signal on the Roche platform and steady or increasing signal on the Abbott platform. It is also possible that the Abbott assay becomes saturated at high antibody concentrations (maximum positive signal ~7.5 S/C), resulting in falsely low "peak" S/C values and preventing the decreasing trend from becoming apparent. Importantly, these trends were not predictive of disease course or admission length as some patients with increasing antibody signals were discharged to home following resolution of symptoms (Patients 1, 3, 5-6, 19) while others with this same pattern remained admitted or were discharged to regional hospitals for continued care (Patients 2 and 4). Similarly, some patients with unchanging or decreasing signal were discharged to home (Patients 7,9,(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(15)(16)18) while others remained admitted or were discharged to regional hospitals (Patients 8,12,17).
A correlation between disease severity and antibody response in patients with PCR-confirmed SARS-CoV-2 infection has been suggested (13,23) and future work will be required to definitively establish this relationship. Anti-SARS-CoV-2 antibody was not detected on at least one assay platform in two convalescent plasma donors with PCR-confirmed infection. Both of these donors exhibited symptoms consistent with SARS-CoV-2 infection. One patient experienced headache, nausea, vomiting, diarrhea, and myalgias but did not experience upper respiratory symptoms or require hospitalization. 38 days after resolution of symptoms, antibody was not detected on the Abbott assay (0.8, cutoff ≥1.4) and was detected but close to the threshold for positivity on the Roche assay (1.9, cutoff ≥1.0). The second patient experienced diarrhea, body aches and prolonged cough but also did not require hospitalization.
Interestingly, this patient's molecular diagnostic test was very weakly positive (cycle threshold One may expect that assays using a total antibody format capable of detecting IgM, IgG and IgA anti-SARS-CoV-2 antibodies would generate positive results sooner than assays that detect IgG only. This was not supported by our study as the Roche (total antibody) assay generated positive results sooner than the Abbott (IgG-specific) assay in one of 18 SARS-CoV-2-positive inpatients with at least one positive sample on both analytic platforms. Similarly, Abbott generated positive results sooner in one of these 18 SARS-CoV-2-positive inpatients. The qualitative results were concordant between the two platforms for the remaining 16 inpatients.
Lastly, our findings confirm previous observations by the assay manufacturers and other independent groups that antibody testing to assess convalescent plasma donors, perform epidemiologic studies or evaluate the cause of a previous uncharacterized respiratory illness should be performed several weeks after resolution of symptoms (24,25). While all immunocompetent hospitalized patients developed detectable anti-SARS-CoV-2 antibodies on both assay platforms at some point in their hospital stay, testing of individuals less than 10 days after the onset of symptoms (patients 1, 7-8, 11, 15, 19) generated antibody results near the detection threshold, resulting in a mixture of negative and weakly positive results.
Several questions remain to be answered during future evaluation of these two assays. As both assays detect antibodies to the viral nucleocapsid rather than spike protein, is either suitable for use in evaluating immune status following infection or immunization? Is one assay preferred for the evaluation of antibody persistence or waning immunity? Should the Roche assay be contraindicated or is it preferred in immunocompromised patients? Do negative serologic test results in patients with PCR-confirmed infection but mild or atypical symptoms suggest the presence of genetic variants that prevent or delay viral entry? Future work should determine whether these assays have a role in clinical laboratory testing beyond the three applications described above.