Clinical Evaluation of the Abbott Alinity SARS-CoV-2 Spike-Specific Quantitative IgG and IgM Assays among Infected, Recovered, and Vaccinated Groups (2024)

Yi-Wei Tang, Editor

Yi-Wei Tang, Cepheid;

Patient samples.

A total of 1,236 individuals (1,428 specimens) were included in this study. This included 263 inpatients with suspected SARS-CoV-2 infection (413 samples); 404 unique patients for specificity and cross-reactivity studies, including 338 patients with samples obtained prior to the emergence of SARS-CoV-2; and 569 individuals (611 samples) tested in outpatient settings with available COVID-19 vaccination information (Fig. 1). For the assessment of clinical sensitivity, only inpatients with suspected SARS-CoV-2 infection with PCR and documented dates of symptom onset were included. In order to accurately evaluate the serological response following vaccination, only outpatients with information concerning their vaccine status were included. The manuscript deals with two aspects, (i) assessing clinical performance of a newly approved quantitation assay, IgG_SP, and (ii) application of this assay, along with IgM_SP and IgG_NC, to evaluate and compare the immune dynamics of SARS-CoV-2 infection and vaccination. This study was approved by the institutional review board of the University of Texas Southwestern Medical Center.

Validation of the new serological assays.

Precision, linearity, sensitivity, specificity, and cross-reactivity assessments were performed to validate the two new serological assays that are designed to semiquantitatively evaluate the presence of IgM_SP and IgG_SP recognizing SARS-CoV-2 SP protein (methods detailed below). The patient samples included those that were collected prior to the COVID-19 pandemic, patient samples with confirmed related respiratory viral infections, multiple myeloma, and COVID-19 samples alongside the vendor-provided quality controls materials. Reverse transcriptase PCR (RT-PCR) testing was used as the predicate/gold standard for assay validation.

Analytical precision.

Precision testing was performed in accordance with Clinical Laboratory Improvement Amendment (CLIA) guidelines and with standard laboratory practices at the University of Texas Southwestern Medical Center. A typical directed 5-day precision study following CLSI EPO5-A3 (5) guidelines was conducted. Two levels of vendor-provided quality controls were tested with 5 replicates in the morning and 5 replicates in the afternoon for 5 days for each test verified.

Measurement range and functional sensitivity.

Linearity (analytical measurement range) testing was performed as a single-day study following CLSI EP06-A (6) . Patient samples with elevated IgG_SP and IgM_SP values (as determined by the IgG_SP and IgM_SP assays described below) were chosen to prepare linearity panels per CLSI EP6-A and according to the manufacturer’s guidelines. Linearity panels consisted of 7 to 8 levels covering the linear range of the test, with one level within the 10th percentile of the lower reportable range, one level within the 90th percentile of the upper reportable range, and one level around the cutoff range of the assays. A minimum of three replicates were tested for each level.

The clinical reportable range was calculated by diluting samples with elevated values that were just below the upper analytical measurement range of the assays at 1:2 and 1:4 dilutions and comparing the results to their neat (undiluted) sample results (≤10% acceptability criteria). Functional sensitivity of the quantitative IgG_SP test (7 arbitrary units [AU]/ml) was verified by running a neat patient sample (8.3 AU/ml) and its 1:1 dilution (expected value, 4.15 AU/ml) 10 times each and calculating their percent coefficient of variation (CV) (acceptable criteria ≤ 20% imprecision) as recommended by the manufacturer.

Carryover and sample stability studies.

Carryover studies were undertaken with a positive (high sample) and negative control (low sample). The high sample was run three times followed by three runs of low sample, and this was repeated three times. Ten patient samples, both SARS-CoV-2 positive and negative, were tested repeatedly over a period of 12 days for specimen stability analysis. An acceptance criterion of ≤10% CV deviation from the baseline/initial results or index values of ≤0.1 for IgM_SP and ≤5 AU/ml for IgG_SP were utilized.

IgM_SP assay.

The AdviseDx SARS-CoV-2 IgM_SP assay has recently been granted emergency use authorization by the U.S. Food and Drug Administration. IgM_SP testing was performed on the Abbott Alinity i platform per the manufacturer’s instructions. The test is a chemiluminescent microparticle (CMIA) assay for semiquantitative assessment of IgM antibodies to the spike protein of SARS-CoV-2 in human serum and plasma sample. A vendor-recommended cutoff of 1.0 (index value) for reactivity/positivity of infection (FDA approval nos. for CoV-2 IgM, 6R87 and H14977R01) was applied.

IgG_SP assay.

AdviseDx SARS-CoV-2 IgG II (FDA approval nos. for CoV-2 IgG II, 6S60 and H18575R01)/SARS-CoV-2 IgG II Quant assay (CE marked) were performed on the Abbott Alinity i platform in accordance with the manufacturer’s package insert. In this antibody CMIA test, the SARS-CoV-2 antigen-coated paramagnetic microparticles bind to the IgG antibodies that attach to the virus’s spike protein in human serum and plasma sample. The resulting chemiluminescence in relative light units (RLU) following the addition of anti-human IgG (mouse, monoclonal) acridinium labeled conjugate in comparison with the IgG II calibrator/standard indicates the strength of response, which reflects the quantity of IgG_SP present. Fifty arbitrary units per milliliter and above in this test are considered positive. This quantitative measurement of IgG_SP can be helpful to evaluate an individual’s humoral response to vaccines.

IgG_NC assay.

The Abbott Alinity i SARS-CoV-2 anti-nucleocapsid protein IgG assay is a semiquantitative CMIA assay that has been previously verified for routine patient testing in our institution’s clinical laboratory, and the index values of 1.4 and above are considered positive (7) per the manufacturer’s instructions.

RT-PCR testing.

The Abbott M2000 or Abbott Alinity m RT-PCR-based molecular testing/confirmation for SARS-CoV-2 were performed using nasopharyngeal specimens collected in viral transport media as previously described (7).

Clinical specificity.

Specificity was evaluated in the pre-COVID-19 era remnant-banked plasma samples from 217 unique patients collected from blood donors from September to November 2019 and early COVID-19 period samples from March to April 2020.

Cross-reactivity studies.

The serum/plasma specimens for cross-reactivity studies included human leukocyte antigen (HLA) lab-confirmed positive samples (collected from 1 January 2015 to 30 September 2019) for cytomegalovirus (CMV) IgG, influenza A/B, respiratory syncytial virus, and endemic human coronaviruses (n = 92). Also, the samples that typically contain significant levels of antibodies, such as lupus patients (n = 29; collected between 2004 and 2007) positive for anti-nuclear antibodies (ANA) and anti-double-stranded DNA and hematological malignancies (HM) (n = 66; collected between March and October 2020), were included.

Statistical analysis.

Data analysis was carried out using GraphPad Prism software (version 9.0.1; San Diego, CA, USA). Data are presented as median with range. When experiments involved more than two groups, one-way analysis of variance (ANOVA) followed by Tukey multiple-comparison post hoc analysis were used to analyze the statistical differences. For experiments involving only two groups, as appropriate, paired or unpaired Student's t tests were performed. A P value of <0.05 was considered statistically significant.

Analytical performance.

For both the IgG_SP and IgM_SP assays, the imprecision was less than 5%. The analytical measurement range of the IgG_SP assay was from 4.2 to 50,000 AU/ml (clinical reportable range, up to 200,000 AU/ml), and for the IgM_SP assay, we observed linearity up to an index value of 16. Manufacturer-recommended functional sensitivity of 7 AU/ml of the quantitative IgG_SP assay was confirmed. There were no carryover issues with both of the assays. The samples kept refrigerated were stable over a 12-day test period.

Clinical specificity and cross-reactivity.

Clinical specificity of the assay was evaluated with serum/plasma from blood donors (SARS-CoV-2 uninfected) obtained before the emergence of SARS-CoV-2. None of these samples were positive (0/217; 100% specificity) for SARS-CoV-2 spike-specific IgG (Fig. 2A) and spike-specific IgM (Fig. 2B) that matched with the manufacturer’s claimed specificity. The median IgG_SP value and IgM_SP index value were found to be 1.92 and 0.05, respectively, well below the corresponding positive cutoff limit of 50 AU/ml for IgG_SP and index value of 1.0 for IgM_SP.

Cross-reactivity of the Abbott SARS-CoV-2 IgG_SP and IgM_SP assays was also evaluated in the setting of respiratory illness and disease conditions where antibody production is significantly elevated (i.e., lupus, hematological malignancies [HM]), primarily multiple myeloma (Fig. 2C and ​andD).D). One hundred twenty-one samples obtained prior to SARS-CoV-2 emergence did not cross-react with IgG_SP. Interestingly, among 66 HM patient samples collected in 2020, 2 patients (∼1.1%) were found to produce a positive value for IgG_SP, indicating some level of cross-reactivity (Fig. 2C). One of these cases (a multiple myeloma patient) was confirmed by PCR as having recovered from COVID-19 upon chart review. The second positive patient was repeatedly negative for SARS-CoV-2 by PCR and strongly positive for hepatitis viral panel serology testing. As no history of SARS-CoV-2 exposure could be established for this patient, further investigation of this IgG_SP-specific apparent cross-reactivity is warranted. No cross-reactivity was observed in the IgM_SP assay (0/187 patients) (Fig. 2D).

Clinical sensitivity.

We evaluated the clinical sensitivity of IgG_SP and IgM_SP assays in our RT-PCR-positive COVID-19 inpatient cohort where reliable information concerning the date of symptom onset and duration was available. Not surprisingly, we noted that the assay sensitivity increased proportionally over time following symptom onset, consistent with the developmental kinetics of a specific antibody response. The sensitivity of IgG_SP was 39% and 58% at 5 and 10 days following symptom onset (Table 1 and Fig. S1A in the supplemental material). Extending the analysis of these patients to 16 to 20 days post-symptom onset and beyond revealed that clinical sensitivity of IgG_SP assay was 96% and 98%, respectively. As expected, clinical sensitivity of IgG_SP assay was much higher (74% within 10 days and 100% within 20 days) based on the RT-PCR-confirmed date of diagnosis (Table S1; Fig. S1B).

Clinical sensitivity of IgM_SP test was comparable to that of the IgG_SP test both within 10 days and 20 days post-symptom onset. At 20 days post-symptom onset and beyond, the sensitivity of the IgM_SP test was 95% (Table 1) and 82% based on RT-PCR-confirmed date of diagnosis (Table S1). Importantly, when IgG_SP and IgM_SP results were combined and analyzed for assay positivity in either format, the sensitivity reached 100% after 15 days from symptom onset and RT-PCR-confirmed date (Table 1 and Table S1).

Longitudinal assessment in COVID-19 inpatients.

We next used IgG_SP values over 360 specimens to assess the change in IgG_SP response over time as a function of days post-COVID-19 RT-PCR testing (DPCR) with the data plotted as a frequency distribution mapping (Fig. S2A). Within the first 15 DPCR, the median IgG_SP values gradually increased and reached an average of 4,466 AU/ml. A dramatic increase in the antibody index value began to occur after 15 days (with a median average of 59,396 AU/ml) (Fig. S2A). Beyond the 27th DPCR, we noticed a decreasing trend in IgG_SP values; however, these results remained above the positivity threshold (≥50 AU/ml). While a lower assay sensitivity was noted in the early period post-symptom onset, it is notable that in some samples, the IgG_SP assay was able to detect positivity within the same time frame as detectable SARS-CoV-2 RNA by RT-PCR. Additionally, the IgG_SP assay was sensitive enough to detect the positivity at day 0, which is the date of infection confirmation by PCR in three samples.

In the same cohort, akin to IgG_SP, we observed the overall tendency of IgM_SP to rise over the positivity threshold of index value of 1.0. Particularly within the first week of symptoms, the distribution was highly inconsistent, and the maximum median index was only 4.345 (Fig. S2B). In contrast, we noted the IgM_SP distribution to progress regularly in tandem over the 2nd week with the median IgM_SP consistently doubling. This subsequently reached as high as ∼38 by the mid-2nd week (17th day). In four samples, IgM_SP was sensitive enough to detect the positivity on day 0 of infection confirmation by PCR (Fig. S2B). While we were able to detect IgM_SP until the 80th day, the IgM_SP values were found to greatly oscillate beyond 23 days with a decreasing trend; however, they were still well above the cutoff index value of 1.0 (Fig. S2B).

IgG_NC assay cutoff for positivity much lower in prior COVID-19 (recovered) patients.

This study included 16 blood samples from known COVID-19-recovered patients (recovered more than 3 to 11 months prior to sampling). Production of NC-specific antibodies is a main differentiator of immune responses to natural SARS-CoV-2 infection versus those to spike-based vaccines. Analysis of this group of patient samples with the IgG_NC assay revealed index values ranging from 0.2 to <1.4 (Table 2; Fig. 3). This is significantly lower than the manufacturer-recommended cutoff index level of ≥1.4 used to detect active (early) infection. However, during waning humoral response (recovery period and postrecovery), our data suggest an index value less than 1.4 and up to 0.2 may predict prior COVID-19 in recovered patients (Table 2; Fig. 3). We therefore applied an index value cutoff below 0.2 for IgG_NC to establish the patients that were truly immunologically naive to SARS-CoV-2 infection (true negatives). This is important in the current setting of increasing numbers of COVID-19-recovered patients in the general population. More reliably, where a combination of tests is possible, the highest diagnostic accuracy or detecting truly negative individuals can be attained when combining an index value of <0.2 for IgG_NC with <50 AU/ml for IgG_SP and index value of <1.0 for IgM_SP. Ninety-nine percent of the samples analyzed using this combination of results were confirmed as negative by RT-PCR or were from patients who were in an early period of infection. In contrast, COVID-19 patients tested between 10 and 20 days post-symptom onset and filtered with the manufacturer-recommended positive cutoff combination of index value of ≥1.4 for IgG_NC with≥50 AU/ml for IgG_SP and an index value of ≥1.0 for IgM_SP showed 99% positivity by PCR (Table 2). Subsequently, we applied these filters wherever we analyzed naive vaccinated and prior COVID-19-vaccinated groups.

SARS-CoV-2 vaccination results in robust SARS-CoV-2 SP-specific antibody responses.

A novel facet of this work is the inclusion of vaccinated patient samples in our analysis. Among those vaccinated, 91% received the Pfizer-BioNTech formulation (n = 132), and 8% obtained the Moderna formulation (n = 13). Antibody responses in the vaccinated group (n = 145) were compared to that of the UV group (n = 424), which included patients that were both RT-PCR positive and negative for SARS-CoV-2 infection. IgG_SP antibody responses ranged from 0 to 49,517 AU/ml, with a median of 6,396 AU/ml (95% confidence interval [CI], 3,814 to 9,729) (Fig. 4A) in the vaccinated group. The median antibody level for vaccinated patients was significantly higher (∼6,400-fold; P < 0.0001) than for UV patients (Fig. 4A). Intriguingly, ∼18% (76/424) of UV patients had IgG_SP values greater than positive detection threshold despite RT-PCR negativity. IgM_SP production was significantly higher (P < 0.0001) in the vaccinated than the UV group, with a more modest median fold increase (∼23%; 1.34 versus 0.06) (Fig. 4B). Nearly 13% (53/424) of UV patients were above the IgM_SP positive threshold despite RT-PCR negativity.

The identification of patients with positive IgG_SP/IgM_SP results despite a lack of vaccination and historically negative RT-PCRs prompted us to investigate IgG_NC levels in these patients as a marker of natural SARS-CoV-2 infection. Surprisingly, about 12% (51/424) were positive (≥1.4), leaving 26 (7%) patients with positive IgG_SP but without IgG_NC positivity. These patients could have silent infection (present or past) because they were also positive for either IgG_SP or IgM_SP. In a majority of UV patients with no known SARS-CoV-2 exposure (82%; 347/424), the IgG_SP and IgM_SP values were determined to be lower than the vendor-recommended cutoff.

Figure S3 illustrates the pattern of IgG_SP antibody value relationships over the days following COVID-19 vaccination (DFD). This group (187 specimens from 145 unique individuals) included both RT-PCR-verified cases and subjects with no exposure to SARS-CoV-2. Within 10 days, the first dose yielded a minimal increase in vaccine-specific antibody response, irrespective of formulation in 2% (4/187) of specimens. More than 54% (102/187) of the vaccinated group had a 5-fold increase, 44% (81/187) had a 10-fold increase, and 17% had a 25-fold increase in the median IgG_SP values compared to the UV group (Fig. S3A). Samples up to 21 days are inclusive of the initial vaccine dose and >21 days are inclusive of the booster. While an increase in value following the first dose of vaccination was clearly observed, not surprisingly, its distribution was variable (Fig. S3A, dark red rectangle). While the number of observations available at or less than 10 days following the first dose was modest, four of the individuals that were tested had values under the positive threshold, and two had values over 45,000 AU/ml. Clearly, after the second dose of vaccination, the IgG_SP values were consistently high and comparatively exhibited a more uniform trend (Fig. S3A, green oval). Interestingly, two patients exhibited a null response following 21 days postvaccination. One of these patients was an HM (multiple myeloma) patient, while the medical history of the other is unknown.

In parallel, SARS-CoV-2-specific IgM_SP levels were also increased following vaccination in naive subjects (Fig. 4B and Fig. S3B). In particular, the distribution analysis demonstrated that following the primary dose of vaccination at week 1 and week 2, ∼71% of the subjects had values above the positive threshold (Fig. S3B). However, at 21 days and beyond post-booster dose, IgM_SP levels again rose, and a noticeable over-the-threshold cluster was seen compared to the primary vaccination (Fig. S3B). Although the number of Moderna-vaccinated recipients in this evaluation is modest, both the IgG_SP and IgM_SP responses appeared to be comparable to those of individuals who received the Pfizer-BioNTech formulation.

Vaccination response in naive population.

Next, we compared the IgG_SP response after the first and booster dose in the naive vaccinated group that was derived by filtering out for IgG_NC of ≥0.2 index cutoff based on Table 2 and Fig. 3. Following the first dose, we observed IgG_SP positivity with a median of 2,217 AU/ml (95% CI, 0 to 44,182), which was drastically increased by 8.2-fold following the booster to a median of 18,272 AU/ml (98% CI, 11,724 to 21,750) (P < 0.001) (Fig. 5A). The cumulative IgM_SP response after the primary dose of vaccination in the naive subjects was found to be with a median index value of 1.1, which is above the positive cutoff (95% CI, 0.87 to 1.4) (Fig. 5B). This was further significantly increased (P = 0.0236) by 1.7-fold to a median of 1.95 (98% CI, 1.16 to 3.3) following the booster dose (Fig. 5B). The IgG_NC remained unchanged between dose 1 and dose 2 in the naive vaccinated group (Fig. 5C). This result suggests that there is a robust response after the booster dose in the naive group.

Vaccination response in prior COVID-19 (recovered) patients.

Currently, the CDC recommends vaccination in those cohorts that are recovered from COVID-19 3 months or prior. Therefore, understanding the immune response in this population is important. The prior COVID-19 group filter was derived from the known recovered patients (Table 2 and Fig. 3). The median IgG_SP values following the first and second doses in the prior COVID-19 group were found to be 17,519 AU/ml (lane 2) and 20,760 AU/ml (lane 3), respectively (Fig. 6A). However, unlike the naive vaccinated first versus second doses (Fig. 5A, lane 1 versus 2), the booster dose in the prior COVID-19 group displayed only a dampened response (Fig. 6A, lane 2 versus lane 3). Similar to the tendency of IgG_SP response between naive vaccinated and prior COVID-19 vaccinated, we noted a blunted and nonsignificant IgM_SP response following booster dose in the prior COVID-19 vaccinated group (Fig. 6B, lane 3 versus lane 2) relative to that observed in the naive vaccinated group (Fig. 5B, lane 2 versus lane 1).

Next, we compared the vaccination response in prior COVID-19 against the immune response in those with active COVID-19 infection (UV_C). The COVID-19 infection status was confirmed by the IgG_NC levels that had a median of 3.3 (96% CI, 2.78 to 4.02), which is 2.4-fold above the positive threshold cutoff (UV_C; Fig. 6C, lane 1). Correspondingly, the median IgG_SP and IgM_SP levels were found to be 1,046 (96% CI, 575.3 to 1,518) and 4.65 (96% CI, 2.78 to 4.02), respectively (Fig. 6A and ​andB,B, lane 1). Both vaccine doses robustly increased the IgG_SP response in relation to the infected group, while it was significant only after dose 1 (P < 0.0001; Fig. 6A, lane 2 versus lane 1). In contrast, IgM_SP levels were significantly lower in the vaccinated group than the infected group (Fig. 6B, lanes 2 and 3 versus lane 1). Interestingly, IgG_NC median values remained much lower (by 0.73) than the positive cutoff of 1.4, suggesting IgG_NC response is infection specific and not vaccination specific. The vaccinated prior COVID-19 groups (primary/first dose [D1] and booster/second dose [D2]) displayed significantly lower IgG_NC response than the unvaccinated COVID-19 group and much below the positive threshold (Fig. 6C, lanes 2 and 3 versus lane 1).

Further examination of antibody levels confirmed the restrained response of booster dose among prior COVID-19-recovered patients (Fig. 7A and ​andB,B, lane 3 versus lane 4) compared to the robust response exhibited by naive vaccinated individuals (Fig. 7A and ​andB,B, lane 1 versus lane 2. Within-patient as well as within-group comparison revealed an unchanged response for IgG_NC (Fig. 7C). As expected, patients with prior COVID-19 exposure exhibited higher IgG_NC levels than those determined to be immunologically naive (Fig. 7C, lanes 3 and 4 versus lanes 1 and 2).

This work was supported by the state of Texas, Tarrant County, Dallas County, the City of Fort Worth, and the city of Dallas.

We thank private philanthropy, including Lyda Hill Philanthropies and the W.W. Caruth, Jr. Fund at Communities Foundation of Texas, for generously funding this research. We also thank Abbott Diagnostics Division (IL, USA) for providing us SARS-CoV-2-specific antibody test kits for the validation of the assays in our facility and further clinical evaluations. We thank Chantale Lacelle, E. Blair Solow, and David Karp for providing their archived samples for specificity and cross-reactivity studies.

Supplemental material is available online only.

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