key: cord-0978211-d3z4vybj
authors: Sibai, Mamdouh; Solis, Daniel; Röltgen, Katharina; Stevens, Bryan A.; Mfuh, Kenji O.; Sahoo, Malaya K.; Shi, Run Z.; Zehnder, James; Boyd, Scott D.; Pinsky, Benjamin A.
title: Evaluation of SARS-CoV-2 Total Antibody Detection via a Lateral Flow Nanoparticle Fluorescence Immunoassay
date: 2021-04-01
journal: J Clin Virol
DOI: 10.1016/j.jcv.2021.104818
sha: eba292b2b31ff5c6f72d4be64a8a568f182fae55
doc_id: 978211
cord_uid: d3z4vybj

BACKGROUND: The coronavirus disease 2019 (COVID-19) endgame may benefit from simple, accurate antibody testing to characterize seroprevalence and immunization coverage. OBJECTIVES: To evaluate the performance of the lateral flow QIAreach anti-SARS-CoV-2 Total rapid nanoparticle fluorescence immunoassay compared to reference isotype-specific IgG, IgM, and IgA SARS-CoV-2 ELISA using S1 or receptor binding domain (RBD) as antigens. STUDY DESIGN: A diagnostic comparison study was carried out using 154 well-characterized heparin plasma samples. Agreement between assays was assessed by overall, positive, and negative percent agreement and Cohen’s kappa coefficient. RESULTS: Overall agreement between the QIAreach anti-SARS-CoV-2 Total and any anti-spike domain (S1 or RBD) antibody isotype was 96.0% (95% CI 89.8 to 98.8), the positive percent agreement was 97.6% (95% CI 91.0 to 99.9), the negative percent agreement was 88.2% (95% CI 64.4 to 98.0). The kappa coefficient was 0.86 (95% CI 0.72 to 0.99). CONCLUSION: The QIAreach anti-SARS-CoV-2 Total rapid antibody test provides comparable performance to high-complexity, laboratory-based ELISA.

Efforts to understand and control the coronavirus disease 2019 (COVID- 19) pandemic have led to the detailed characterization of the humoral response to SARS-CoV-2 infection. At a median of approximately 2 weeks after onset of symptoms, specific IgM, IgG and IgA antibodies become detectable in blood [1, 2] . Antibody titers peak at around 1 month post symptom onset, and then decrease, relatively rapidly for IgM and IgA, and more gradually for IgG [3] . In vaccine licensing studies, SARS-CoV-2 immunization elicits robust antibody responses and at least short-term protection from natural infection [4] [5] [6] .

SARS-CoV-2 antibody testing is recommended for the evaluation of patients with a high clinical suspicion of infection and repeatedly negative nucleic acid amplification tests, as well as in the assessment of suspected multisystem inflammatory syndrome in children [7, 8] . SARS-CoV-2 antibody testing is also a critical public health tool, enabling surveillance efforts to characterize seroprevalence and vaccine coverage.

Methods for SARS-CoV-2 antibody detection target various viral antigens and include laboratory-based testing, such as enzyme-linked immunosorbent assays (ELISA), as well as rapid, lateral flow immunoassays (LFIA) that also may be used at the point-of-care. These rapid assays provide a low-throughput antibody testing option for laboratories with limited resources and are particularly useful for epidemiologic field studies. However, a meta-analysis evaluating the diagnostic accuracy of SARS-CoV-2 serologic testing concluded that LFIAs were consistently less sensitive than ELISA or CLIA methods [9] , and subsequent studies have reported a wide range of sensitivities and specificities [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] . Nevertheless, the Infectious J o u r n a l P r e -p r o o f Diseases Society of America (IDSA) recommends against the use of IgG or IgM antibody combination tests, where detecting either antibody isotype is used to define a positive result [7] .

Notably, of the LFIAs that have obtained FDA emergency use authorization, 87.5% (14/16) are combination tests [25] .

In this study, well-characterized clinical plasma specimens were utilized to evaluate a SARS-CoV-2 total antibody (IgG, IgM, IgA) nanoparticle fluorescence immunoassay (QIAreach anti-SARS-CoV-2 Total) that uses as antigen the spike protein S1 domain, which also contains the angiotensin converting enzyme-2 (ACE2) receptor binding domain (RBD).

This study was approved by the Stanford Institutional Review board (IRB protocol #48973). Per IRB assessment, informed consent was waived for this study.

Isotype-specific IgG, IgM, and IgA SARS-CoV-2 S1 and RBD ELISAs were performed manually as previously described, as was a competition ELISA to detect antibodies blocking binding of the ACE2 to RBD [1] . Pre-pandemic samples were tested using automated versions of the SARS-CoV-2 RBD IgG and IgM ELISAs on the Quanta-Lyser ESP600 (Innova Diagnostics, Inc. San Diego, CA).

Archived heparin plasma samples (n=100) collected from fifty-eight SARS-CoV-2 reverse transcriptionpolymerase chain reaction (RT-PCR) positive patients and tested by isotypespecific IgG, IgM, and IgA SARS-CoV-2 S1 and RBD manual ELISAs as well as the RBD-ACE2 blocking assay, were selected to encompass a range of OD values, patterns of isotype reactivity, and blocking activity. Pre-pandemic heparin plasma samples (n=42) negative by automated SARS-CoV-2 RBD IgG and IgM ELISAs were used to evaluate specificity. S1 and RBD IgG, IgA, and IgM negative heparin plasma samples (n=12) with IgM plastic binding activity were also included in specificity experiments.

QIAreach anti-SARS-CoV-2 Total Test (Qiagen, Germantown, MD) was performed according to the manufacturer's instructions. Briefly, the Access eHub was connected to a power source via USB. Next, a Processing Tube and eStick were inserted into the eHub. 300 µL of Diluent Buffer was transferred into the Processing Tube, followed by 50 µL of the heparin plasma sample.

Using a pipette set to 150 µL, the sample was mixed at least 4 times in the Processing Tube, and then 150 µL of the mixture was added to the eStick. A result displayed within 3 to 10 minutes (180 to 600 seconds). Time to result in seconds was recorded.

Overall percent agreement, positive percent agreement (PPA), negative percent agreement (NPA), and Cohen's kappa coefficient with associated 95% confidence intervals (95% CI) were calculated using GraphPad online. Cohen's kappa values were interpreted according to Landis and Koch [26] . (Table 1) .

Overall percent agreement between the QIAreach Total and ELISAs detecting any anti-S1antibody isotype was 91.0% (95% CI 83.6 to 95.4). The PPA was 98.7% (95% CI 92.2 to 100.0) and the NPA was 66.7% (95% CI 46.6 to 82.2). The kappa coefficient was 0.73 (95% CI 0.56 to 0.89), indicating substantial agreement (Table 2 ). When the QIAreach Total was compared with specific anti-S1 antibody isotypes (IgG, IgM, IgA) the NPA ranged from 29.8 to 57.1%. Similar performance was observed when the QIAreach Total was compared to the anti-RBD ELISAs (Table 2 ).

The performance of the QIAreach Total was then assessed using detection of any anti-spike domain (S1 or RBD) antibody isotype as reference. The overall percent agreement was 96.0% (95% CI 89. 8 Figure S1 ). This analysis revealed four discrepant results (Table S1) . Future work will be required to directly compare the QIAreach Total to other LFIAs, and to assess its performance at the point-of-care with finger-stick blood specimens.

The transition to point-of-care testing should be straightforward as the QIAreach Total is simple to perform, with minimal hands-on-time. Whereas conventional LFIAs demonstrate faint banding that may be difficult to interpret and is subject to low inter-rater reliability [14, 21, 23] , QIAreach Total provides digital qualitative results without the requirement for visual interpretation of band reactivity. The results are also available rapidly; the QIAreach Total exhibited a median time to result of 3 minutes and 10 seconds. The time to result, however, is not linearly associated with ELISA OD values and the test, in its current design, should not be used in a semi-quantitative manner.

The major strength of this study is the use of a sample set comprised of a range of ELISA OD Table 1 

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PPA, positive percent agreement; NPA, negative percent agreement; S1, spike protein S1 domain; RBD, spike protein receptor binding domain