Skip to main content
PMC home page
mSphere logoLink to mSphere
. 2022 Jun 15;7(4):e00193-22. doi: 10.1128/msphere.00193-22

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization

Amy B Karger a,✉, James D Brien b, Jayne M Christen c, Santosh Dhakal d, Troy J Kemp c, Sabra L Klein d, Ligia A Pinto c, Lakshmanane Premkumar e, John D Roback f, Raquel A Binder g, Karl W Boehme h, Suresh Boppana i,j, Carlos Cordon-Cardo k, James M Crawford l, John L Daiss m, Alan P Dupuis II n, Ana M Espino o, Adolfo Firpo-Betancourt k, Catherine Forconi g, J Craig Forrest h, Roxie C Girardin n, Douglas A Granger p, Steve W Granger p, Natalie S Haddad q, Christopher D Heaney r, Danielle T Hunt n, Joshua L Kennedy s,t,u, Christopher L King v, Florian Krammer w, Kate Kruczynski r, Joshua LaBaer x, F Eun-Hyung Lee q, William T Lee n,y, Shan-Lu Liu z,aa,bb,cc, Gerard Lozanski dd, Todd Lucas ee,ff, Damodara Rao Mendu k, Ann M Moormann g, Vel Murugan x, Nkemakonam C Okoye l, Petraleigh Pantoja gg,*, Anne F Payne n, Jin Park x, Swetha Pinninti i, Amelia K Pinto b, Nora Pisanic r, Ji Qiu x, Carlos A Sariol gg,hh, Viviana Simon w, Lusheng Song x, Tara L Steffen b, E Taylor Stone b, Linda M Styer n,y, Mehul S Suthar ii,jj,kk,ll, Stefani N Thomas a, Bharat Thyagarajan a, Ania Wajnberg mm, Jennifer L Yates n,y, Kimia Sobhani nn
Editor: Michael J Imperialeao
PMCID: PMC9429934  PMID: 35703544

ABSTRACT

In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and “to develop, validate, improve, and implement serological testing and associated technologies” (https://www.cancer.gov/research/key-initiatives/covid-19/coronavirus-research-initiatives/serological-sciences-network). SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology standard reference material and first WHO international standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. SeroNet institutions reported development of a total of 27 enzyme-linked immunosorbent assay (ELISA) methods, 13 multiplex assays, and 9 neutralization assays and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. In conclusion, SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons.

IMPORTANCE SeroNet institutions have developed or implemented 61 diverse COVID-19 serological assays and are collaboratively working to harmonize these assays using reference materials to establish standardized reporting units. This will facilitate clinical interpretation of serology results and cross-comparison of research data.

KEYWORDS: COVID-19, SeroNet, assay harmonization, serology

INTRODUCTION

The National Cancer Institute (NCI) Serological Sciences Network for COVID-19 (SeroNet) was launched on 8 October 2020 as a collaborative initiative to expand research on immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SeroNet is comprised of investigators from 25 U.S. biomedical research institutions, working in partnership with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center, which is managed by FNLCR (1). Of the 25 participating research institutions, 8 are designated as Serological Sciences Centers of Excellence (funded by U54 grants), 13 are funded with U01 grants to carry out specific research projects related to COVID-19 immunity, and 4 institutions are funded by subcontracts and are designated as Serological Sciences Network Capacity Building Centers (1).

One of the primary goals of this partnership is “to develop, validate, improve, and implement serological testing and associated technologies” (1). To this end, SeroNet formed a working group, the Serology Assays, Samples, and Materials Operations Group (abbreviated as Serology Assay Ops), in December 2020 to allow for coordinated development and collaborative sharing of serology assay procedures and to establish processes for harmonizing and standardizing methodologies using reference materials across institutions. Establishing harmonized and standardized SARS-CoV-2 serological assays can allow cross-comparison and pooling of research study results and facilitate clinical interpretation of results for patient care.

While there are 85 serological assays approved by the FDA for emergency use (2), the quick development of assays has led to the lack of harmonized cutoffs and reporting units. Furthermore, there are no consensus guidelines on reporting standards or clarity on the clinical interpretation and relevance of results. This has created a complex landscape for interpreting both research and clinical serological assay results. For example, several studies have reported on heterogeneity in serological assay performance that would have a significant impact on research study conclusions and clinical interpretations related to longitudinal serosurveillance (3–6). Specifically, certain assays demonstrate reduced sensitivity over time after an initial SARS-CoV-2 infection diagnosis. Muecksch et al. reported that the Abbott SARS-CoV-2 anti-nucleocapsid IgG assay dropped from a peak sensitivity of 98% at 21 to 40 days post-PCR diagnosis to around 70% when patients were tested ≥81 days postdiagnosis, whereas the Roche Elecsys SARS-CoV-2 anti-nucleocapsid total antibody assay and Siemens SARS-CoV-2 anti-receptor-binding domain (anti-RBD) total antibody assay both maintained high sensitivity (95 to 100%) on the same set of serial samples (3). Narowski et al. also found a significant decline in the longitudinal sensitivity of their lab-developed nucleocapsid assay in a study of health care workers (6). Perez-Saez et al. similarly demonstrated that the rates of seroreversion at least 8 months after the initial infection differed greatly depending on the serological assay used (4). While the seroreversion rate of the EuroImmun semiquantitative anti-S1 IgG enzyme-linked immunosorbent assay (ELISA) was 26%, the rates were significantly lower for the Roche anti-nucleocapsid total antibody assay (1.2%) and the Roche semiquantitative anti-RBD total antibody assay (0%) (4). Additionally, numerous studies rely on neutralization assays as gold standard methods for determining the functional relevance of ligand-binding methods, but comparison studies have demonstrated variability in results for live-virus neutralization, pseudovirus neutralization, and surrogate neutralization assays (e.g., ACE2 inhibition assays) (7–9), raising the importance of assay harmonization and standardization across laboratories.

Therefore, SeroNet aims to address these knowledge gaps in SARS-CoV-2 serological assay research by establishing collaborative initiatives to characterize, compare, and harmonize SARS-CoV-2 serological assays. This paper describes the depth and breadth of serological assays developed and implemented within the SeroNet consortium and outlines a proposed process to establish assay traceability to the U.S. SARS-CoV-2 serology standard reference material and to the WHO international standard (IS; 20/136) for these diverse assays, with the ultimate goal of establishing harmonized reporting standards calibrated to the international standard. Availability of both national and international standards is crucial to provide easy accessibility to end users and due to the limited volume of international standard available; all national standards should be calibrated to the international standard to provide harmonized traceability. These collaborative efforts will facilitate cross-comparison of results and provide clarity for their clinical interpretation, including in response to circulating SARS-CoV-2 variants.

RESULTS

SeroNet serology assay data.

Of the 25 institutions involved with SeroNet, 23 reported performing between one and seven serology assays and provided descriptive and performance data. Serology assay data were also obtained from the Frederick National Laboratory for Cancer Research (FNLCR) and National Institute of Standards and Technology (NIST), both of which collaborate with SeroNet. Collectively, SeroNet institutions reported development of 27 in-house ELISA methods (Table 1) (6, 10–26). The majority of ELISA methods were developed for testing of serum and/or plasma, with additional methods available for testing dried blood spots (DBS), saliva/oral fluid, and breast milk. Two methods have been granted FDA emergency use authorization (EUA), 3 methods are pending FDA EUA, 4 methods are validated for high-complexity testing in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory, and 18 methods are for research use only (RUO). Diagnostic sensitivity and specificity for in-house ELISA methods range from 67.4 to 100% and 90 to 100%, respectively.

TABLE 1.

Laboratory-developed singleplex ELISAsa

Sample type(s) Antigen(s) Isotype Result type Assay sensitivity and specificity Center/institution Reference(s) Regulatory status
Serum, plasma, dried plasma RBD IgG (IgA/IgM being eval) Qualitative Day 0–7 after infection: sensitivity, 73.01%. Day 8–14 after infection: sensitivity, 100%. Day >15 after infection: sensitivity, 100%; specificity (n = 388 samples collected prior to COVID-19 pandemic), 97.68%. Emory University 21 FDA EUA granted
Serum, plasma RBD and Spike IgG, IgM, IgA Semiquantitative Sensitivity, 95%; specificity, 100% (n = 38 positive, n = 74 negative sera tested) Mount Sinai 12, 19, 20 FDA EUA granted
Serum, plasma, saliva RBD Total Ig, with IgG, IgM, IgA titers Qualitative Overall sensitivity, 82.5%; overall specificity, 100% (n = 300). At >14 days from symptom onset, sensitivity, 100%, and specificity, 100% (n = 261). University of Minnesota 18, 22 Assays validated in a high-complexity-testing CLIA laboratory
Serum, plasma RBD IgG, IgM Qualitative Sensitivity, 91% for RBD IgG 15–21 days post-onset of symptoms, 100% >21 days post-onset of symptoms, 90% for RBD IgM 15–21 days post-onset of symptoms, and 100% >21 days post-onset of symptoms; specificity, 99.75% for RBD IgG and 100% for RBD IgM Stanford University 10 Assays validated in a high-complexity-testing CLIA laboratory
Serum, plasma RBD-ACE2 Total IgG that blocks RBD-ACE2 binding Semiquantitative NA; used as a follow-up assay in seropositive specimens Stanford University 10 Assay validated in a high-complexity-testing CLIA laboratory
Serum, plasma RBD IgG, IgM + IgG Quantitative (IgG); qualitative (IgM + IgG) Sensitivity, 98% (n = 181); specificity, 98.9% (n = 181). University of Puerto Rico 25, 53 Assay validated in a high-complexity-testing CLIA laboratory
Serum, plasma Spike IgG Quantitative Sensitivity, 98.3% (n = 60); specificity, 99.3% (n = 150) Frederick National Laboratory NR RUO
Serum, plasma Spike IgM Quantitative Sensitivity, 93.8% (n = 30); specificity, 97.6% (n = 80) Frederick National Laboratory NR RUO
Serum, plasma Nucleocapsid IgG Quantitative Sensitivity, 97% (n = 34); specificity, 100% (n = 99) Frederick National Laboratory NR RUO
Serum, plasma Nucleocapsid IgM Quantitative NR Frederick National Laboratory NR RUO
Serum, plasma, saliva RBD Total Ig Qualitative Sensitivity, 95% (n = 259; 9 or more days after symptom onset), specificity, 96% (n = 535) University of North Carolina 6, 16 FDA EUA pending
Serum, plasma, saliva Spike NTD Total Ig Qualitative Sensitivity, 92% (n = 259; 9 or more days after symptom onset), specificity, 94% (n = 535) University of North Carolina 6 FDA EUA pending
Serum Spike, RBD IgG Semiquantitative NR CVVR/BIDMC/Harvard 11 RUO
Serum, plasma, breast milk RBD IgG, IgA, IgM Semiquantitative NR CVVR/BIDMC/Harvard 14, 23 RUO
Serum, plasma Spike IgG Quantitative Sensitivity, 100%; specificity, 98.8% Tulane University NR RUO
Serum, plasma RBD IgG Quantitative NR Tulane University NR RUO
Serum, plasma Nucleocapsid IgG Quantitative NR Tulane University NR RUO
Plasma, serum Spike, RBD IgM, IgG, IgA Semiquantitative Spike: IgG, sensitivity, 96.6%, and specificity, 96.7%); IgA, sensitivity, 99.3%, and specificity, 90%; IgM, sensitivity, 97.9%, and specificity, 100%. RBD: IgG, sensitivity, 97.3%, and specificity, 100%; IgA, sensitivity, 99.3%, and specificity, 96.7%; IgM, sensitivity, 97.9%, and specificity, 96.7%. IgG data based on 126 convalescent plasma donors and 30 prepandemic samples; IgM/IgA data based on 20 hospitalized donors and 30 prepandemic samples. Johns Hopkins University 15 RUO
Serum, plasma Spike (ECD), RBD IgG Semiquantitative NR University of Texas-Austin 17 RUO
Serum, plasma RBD IgG Qualitative Sensitivity, 100% (n = 155); specificity, 96.5% (n = 133) Arizona State University NR RUO
Serum, DBS RBD IgG, IgM Qualitative Sensitivity, 97% (n = 39); specificity, 100% (n = 37) University of Arkansas for Medical Sciences 54 RUO
Serum, DBS RBD, spike, nucleocapsid IgG, IgM Qualitative Sensitivity, 97% (n = 39); specificity, 100% (n = 37) University of Arkansas for Medical Sciences 13 RUO
Serum, plasma, breast milk RBD, spike, nucleocapsid IgG, IgM, IgA Quantitative (IgG); Qualitative (IgM, IgA) Sensitivity, 97% (n = 114); specificity, 99% University of Alabama—Birmingham NR RUO
Serum, plasma RBD, nucleocapsid, spike trimer IgG, IgA Quantitative RBD: sensitivity, 70.9% for IgG and 74.4% for IgA; specificity, 100% for both IgG and IgA. Nucleocapsid: sensitivity, 81.4% for IgG and 77.9% for IgA; specificity, 98.5% for IgG and 100% for IgA). Spike trimer: sensitivity, 67.4% for both IgG and IgA; specificity, 98.5% for IgG and 100% for IgA. Data based on PCR-confirmed COVID-19 hospitalized patients (n = 86) and negative prepandemic samples (n = 65). University of Massachusetts Chan Medical School 26 RUO
Serum, Plasma Nucleocapsid IgG Qualitative Sensitivity, 100% (n = 44); specificity, 99.5% (n = 202) The Ohio State University 24 FDA EUA pending
Serum Nucleocapsid IgG Qualitative NR The Ohio State University NR RUO
Oral fluid Nucleocapsid IgG Qualitative Sensitivity, 92% (n = 24); specificity, 98% (n = 85) Salimetrics NR RUO
Open in a new tab
a

ACE2, angiotensin-converting enzyme 2; BIDMC, Beth Israel Deaconess Medical Center; CLIA, Clinical Laboratory Improvement Amendments; CVVR, Center for Virology and Vaccine Research; DBS, dried blood spots; ECD, extracellular domain; EUA, emergency use authorization; FDA, Food and Drug Administration; NA, not applicable; NR, not reported; NTD, N-terminal domain; RBD, receptor-binding domain; RUO, research use only.

Eight institutions reported development or use of multiplex or protein arrays for antibody detection (Table 2) (27–37). Sample types include serum, plasma, DBS, saliva, and bronchoalveolar lavage (BAL) fluid. Diagnostic sensitivity and specificity for multiplex and protein array methods range from 85 to 98.8% and 95.2 to 100%, respectively. Neutralization assays were developed by 9 institutions, with sample types including serum, plasma, BAL fluid, nasal wash, DBS, and breast milk (Table 3) (15, 24, 29, 38–50). Assays fall into three mechanistic categories: competitive binding assays, pseudotyped neutralization assays, and live-virus neutralization assays. The competitive binding assay measures the ability of antibodies to block interactions between the SARS-CoV-2 receptor-binding domain and human ACE2 receptor. Virus pseudotype neutralization assays, mainly HIV and vesicular stomatitis virus (VSV) based, use full-length spike incorporated in the viral particle to measure the capability of neutralizing antibodies to block viral entry into the target cells. SARS-CoV-2 live-virus plaque or focus reduction neutralization assays measure the ability of neutralizing antibodies to block the spreading infection of authentic SARS-COV-2 in cell culture. Diagnostic sensitivity and specificity for neutralization methods developed within SeroNet range from 93 to 100% and 97 to 100%, respectively. Lastly, 9 institutions report use of 12 commercial serology methods (Table 4). Commercial methods detect IgG, IgM, and/or total Ig to spike, RBD, and/or nucleocapsid antigens in serum or plasma. Of the commercial methods in use, 10 have FDA EUA, 1 is pending FDA EUA, and 1 is RUO.

TABLE 2.

Laboratory-developed multiplex assaysa

Sample type(s) Antigen(s) Isotype Result type Assay sensitivity and specificity Center/institution Reference(s) Regulatory status
DBS, serum Spike S1, nucleocapsid IgG Qualitative Sensitivity, DBS, 94% for symptomatic (n = 774 samples collected >20 days after PCR+ result) and 85% for asymptomatic (n = 115 samples collected >20 days after PCR+ result); specificity, DBS, 99% (n = 730), and serum, 99% (n = 701) Wadsworth 27, 28 NYS CLEP approved
Serum, plasma, DBS Spike, nucleocapsid, RBD Total Ig Semiquantitative Sensitivity, >97%; specificity, 99% Wadsworth 29 FDA EUA granted; NYS CLEP approved
Serum, plasma, DBS Spike, nucleocapsid, RBD IgG, IgM, IgA Semiquantitative Sensitivity, >97%; specificity, 99% Wadsworth 30 NYS CLEP approved; FDA EUA pending
Oral fluid, serum, plasma Spike, RBD, nucleocapsid IgG, IgM, IgA Semiquantitative Oral fluid IgG assay, sensitivity, 98.8% ≥15 days post-symptom onset (n = 81); specificity, 100% (n = 127) Johns Hopkins University, supporting Michigan State University 31, 36 Oral fluid assays validated in a high-complexity-testing CLIA laboratory; serum/plasma RUO
Serum, plasma, BAL, DBS Spike, RBD (different variants), nucleocapsid IgG Quantitative Sensitivity, >97% (n = 89); specificity, 99% (n = 260) Case Western Reserve University 32 RUO
Serum, plasma, saliva, BAL fluid Spike, RBD, nucleocapsid IgA Quantitative Sensitivity, >98%; specificity, 99% Case Western Reserve University 32 RUO
Serum, plasma Spike IgG Quantitative Sensitivity, ≥93%; specificity, 100% NIST 33 RUO
Serum, plasma RBD IgG Semiquantitative Sensitivity, ≥93%; specificity, 100% NIST 33 RUO
Serum, plasma RBD, nucleocapsid IgG Semiquantitative Nucleocapsid: sensitivity, 90.3% (n = 155), and specificity, 98.0% (n = 133). RBD: sensitivity, 90.1% (n = 155), and specificity, 97.0% (n = 133). Arizona State University NR FDA EUA pending
Serum Spike, nucleocapsid, RBD IgG, IgM, IgA Quantitative NR Yale 34 RUO
Serum Alpha, Beta, Gamma, and Delta variants (spike, RBD) IgG, IgM, IgA Quantitative NR Yale 35 RUO
Saliva Spike, nucleocapsid, RBD IgG Semiquantitative Sensitivity: nucleocapsid, 97.7%, RBD, 92.9%, and spike, 98.8%. Specificity: nucleocapsid, 95.2%, RBD, 96.4%, and spike, 97.6%. Combined nucleocapsid and spike sensitivity, 96.5%, and specificity, 98.8%. Salimetrics NR RUO
Serum, plasma Spike S1, S1-RBD, nucleocapsid, S1-NTD IgG, IgA, IgM (combined); IgG, IgA, IgM (individual) Quantitative Sensitivity: combined antigens and isotypes, 99%; S1-RBD combined isotypes, 99%, and S1-RBD IgG, 99%. Specificity: combined antigens and isotypes, 99%, S1-RBD combined isotypes, 99%, and S1-RBD IgG, 99%. During the acute phase, sensitivity, 92%, and specificity 99%. Emory/MicroB-plex 37 RUO
Open in a new tab
a

BAL, bronchoalveolar lavage; CLIA, Clinical Laboratory Improvement Amendments; NIST, National Institute of Standards and Technology; NYS CLEP, New York State Clinical Laboratory Evaluation Program.

TABLE 3.

Neutralization assaysa

Sample type(s) Antibody neutralization assay type Result type Assay sensitivity and specificity Center/institution Reference(s) Regulatory status
Serum, plasma, BAL fluid HIV lentiviral vector Quantitative Sensitivity, 100%, and specificity, 100%, using SeroNet FNLCR blinded reference panel set (n = 110) The Ohio State University 24 RUO
Serum, plasma Live-virus neutralization assay (microneutralization) Semiquantitative NR Mount Sinai 38, 39 RUO
Serum, plasma, BAL fluid Live-virus neutralization assay (FRNT) Quantitative Sensitivity, 93%; specificity, 100% Saint Louis University 25, 40 RUO
Serum, plasma, BAL fluid Live-virus neutralization assay (FRNT/FRNT-mNG/PRNT) Quantitative NR Emory 41 RUO
Serum, plasma, DBS Live-virus neutralization assay (PRNT) Qualitative PRNT50: sensitivity, 100%; specificity, 97%. PRNT90: sensitivity, 97%; specificity, 100% Wadsworth 29, 42 NYS CLEP approved (serum and plasma)
Serum, plasma, breast milk VSV pseudotype particle-based assay Quantitative NR University of Alabama—Birmingham NR RUO
Serum, plasma, nasal washes TCID50 neutralization assay Semiquantitative NR Johns Hopkins University 15, 43–47 RUO
Serum, plasma ACE2 competitive binding assay Quantitative Sensitivity, 93.8%; specificity, 99.4% University of Puerto Rico 25 RUO
Serum, plasma Lentivirus-based pseudovirus assay for Wuhan D614G, Brazil, South Africa, and Delta variants. Assay performed in CHO/ACE2 cells. Quantitative Sensitivity, 100%; specificity, 100% Tulane University 50 RUO
Open in a new tab
a

CHO, Chinese hamster ovary; FNLCR, Frederick National Laboratory for Cancer Research; FRNT, focus reduction neutralization test; HIV, human immunodeficiency virus; mNG, mNeonGreen; PRNT50 and PRNT90, 50% and 90% plaque reduction neutralization test; TCID50, 50% tissue culture infectious dose; VSV, vesicular stomatitis virus.

TABLE 4.

Commercial assays

Instrument/assay Antigen(s) Isotype Result type Center/institution Regulatory status
Abbott Alinity Spike IgM Semiquantitative Mount Sinai FDA EUA granted
Abbott Architect Spike IgG Semiquantitative Cedars-Sinaia FDA EUA granted
Abbott Architect Nucleocapsid IgG Qualitative Cedars-Sinaia FDA EUA granted
Beckman Coulter Access Spike IgG Semiquantitative Arizona State University FDA EUA granted
Beckman Coulter Access Spike IgM Qualitative Arizona State University FDA EUA granted
DiaSorin Liaison Spike IgG Qualitative (Feinstein/Northwell, Kaiser); quantitative (The Ohio State University) Feinstein/Northwell, Kaiser, The Ohio State University FDA EUA granted
DiaSorin Liaison Spike IgM Qualitative Feinstein/Northwell FDA EUA granted
Kantaro SeroKlir Spike, RBD IgG Semiquantitative Mount Sinai FDA EUA granted
Kantaro quantitative SARS-CoV-2 Spike, RBD IgG Quantitative Mount Sinai FDA EUA pending
Meso Scale Discovery Spike, nucleocapsid IgG, IgM Quantitative University of Alabama—Birmingham, CVVR/BIDMC/Harvard, Johns Hopkins University, Stanford RUO
Roche Elecsys anti-SARS-CoV-2 Nucleocapsid Total Ig Qualitative University of Minnesota, Feinstein/Northwell FDA EUA granted
Roche Elecsys anti-SARS-CoV-2 S RBD Total Ig Semiquantitative University of Minnesota, Feinstein/Northwell FDA EUA granted
Siemens Atellica Spike Total Ig Semiquantitative Kaiser, The Ohio State University FDA EUA granted
Open in a new tab
a

Samples sent to Abbott Diagnostics for testing.

Establishment of SeroNet assay traceability to the U.S. SARS-CoV- 2 serology standard and first WHO international standard for anti-SARS-CoV-2 immunoglobulin.

Units for the U.S. SARS-CoV-2 serology standard were initially established by FNLCR based on measurements performed by eight laboratories (Table 5). Subsequently, FNLCR further established traceability of the U.S. SARS-CoV-2 serology standard to WHO IS 20/136 by using four FNLCR ligand binding serology assays, with assessment of neutralization tested at NIAID’s Integrated Research Facility (IRF) (Table 5). The U.S. SARS-CoV-2 serology standard was made available to the public in December 2020. Thus, far, there have been 124 requests for U.S. SARS-CoV-2 standard material and 19 requests for the reference panel samples.

TABLE 5.

Units assigned to the U.S. SARS-CoV-2 serology standarda

Units assigned by FNLCR
 WHO-calibrated units
Functional activity Spike and nucleocapsid IgM Spike and nucleocapsid IgG Functional activity Spike IgG Nucleocapsid IgG Spike IgM Nucleocapsid IgM
200 NU/mL 100 BAU/mLb 1200 BAU/mLb 815 IU/mL 764 BAU/mLc 681 BAU/mLc 246 BAU/mLc 1037 BAU/mLc
Open in a new tab
a

WHO, World Health Organization; NU, neutralizing units; IU, international units.

b

BAU/mL, binding assay units per milliliter.

c

BAU/mL, binding antibody units per milliliter.

DISCUSSION

SeroNet has collectively established a diverse array of methodologies for measurement of SARS-CoV-2 antibodies in a variety of biological fluids. Methods include laboratory-developed ELISAs, multiplex assays, and neutralization assays, most used for research-only purposes, as well as commercial assays available for patient care or research studies. Assays have been developed to test unique sample types, including DBS, saliva/oral fluid, breast milk, nasal washes, and bronchoalveolar lavage fluid. Binding assays identify IgM, IgG, IgA, and/or total antibodies to nucleocapsid, spike, RBD, and/or N-terminal domain (NTD) antigens, and neutralization assays rely on three methods to quantify antibodies with functional neutralizing activity. Assays vary in result reporting, with qualitative, semiquantitative, and quantitative assays. This diversity of assay methods allows for robust investigation of multiple aspects of the serological response to SARS-CoV-2 infection and vaccination and for cross-comparison of assay performance across platforms and institutions within SeroNet.

With the rapid development of numerous methods for serological assessment, as exemplified by the depth and breadth of assays within SeroNet, it is critical to establish assay harmonization and standardized reporting units to facilitate cross-comparison of results across studies, as well as for streamlined meta-analyses. To this end, FNLCR has provided the U.S. SARS-CoV-2 serology standard reference material, which has traceability to the first WHO international standard for anti-SARS-CoV-2 immunoglobulin, to SeroNet sites performing serological assays, to allow establishment of standardized reporting of semiquantitative or quantitative results in binding antibody units (BAU) per milliliter traceable to the WHO standard. For qualitative assays, standardization is crucial for comparing and then harmonizing assay cutoffs for positivity that are traceable to the WHO standard. These efforts may more rapidly facilitate the establishment of a universal cutoff as a correlate of protection, which will be critical to broaden the clinical utility of serological testing for patient care, will allow vaccine trials to transition to an immunogenicity endpoint rather than morbidity or mortality endpoints (immunobridging), and will guide decisions regarding optimal scheduling of future vaccine doses to optimize protective efficacy for the general immunocompetent population and susceptible immunocompromised subpopulations.

While the first step toward harmonization is calibration of assays to a common standard, there will be remaining challenges to pooling data given differences in assay performance metrics, sample types, isotypes, result type (qualitative, semiquantitative, or quantitative), methodologies, and antigen targets. SeroNet and FNLCR continue to work collaboratively to lay the groundwork for effective serology assay data pooling; FNLCR is currently conducting a comprehensive assay comparison study using split blinded samples sent to different SeroNet laboratories to assess the success of harmonization efforts, as well as assay performance (repeatability, sensitivity, and specificity). Currently, there is a broad range of epidemiologic studies being conducted across SeroNet, as previously described, and SeroNet’s future work will include a focus on integrated analysis of pooled data with standardization of reported data elements and assay harmonization (51).

In summary, SeroNet is well positioned to rapidly and collaboratively advance our understanding of the immune response to both SARS-CoV-2 infection and vaccination, with ongoing evaluation of serological responses to SARS-CoV-2 variants of concern. The collective effort of institutions involved with SeroNet, to both establish diverse and complementary serological assays and establish traceability of these diverse assays to the WHO standard, will allow for comprehensive investigation of immune responses and facilitate pooled analyses within the SeroNet consortium. This will enable achievement of the ultimate goal: establishment of a universal correlate-of-protection cutoff, which will provide a foundation for broader clinical use of serological testing, as a guide for future decisions on scheduling of COVID-19 vaccine boosters, as well as for general assessment of COVID-19 vaccine immune responses against vaccine viruses and newly evolving variants of concern.

MATERIALS AND METHODS

Compilation of data on SeroNet serological assays.

SeroNet institutions were queried by email between January and July 2021 and asked to complete a comprehensive serological assay survey to describe serological assays developed or implemented at the institutions. The survey requested information on assay and sample type(s), instrument platform and reagents, data output, antibody isotype(s) detected, targeted antigens and virus strain(s), assay performance, cutoffs, use of standards and quality controls, method comparison studies, regulatory status, current use/applications for assays, and publications using each assay.

Protocol for establishing traceability of serology assays to the U.S. SARS-CoV-2 serology standard and first WHO international standard for anti-SARS-CoV-2 immunoglobulin.

FNLCR developed a protocol for SeroNet institutions to establish serology assay traceability to the U.S. SARS-CoV-2 Serology Standard. Through FNLCR’s participation in the drafting group for the WHO Manual for the Preparation of Reference Materials for Use as Secondary Standards in Antibody Testing, the protocol has been made available to the public as of 11 May 2022 (see Appendix 8 of reference 52).

In short, for enzyme-linked immunosorbent assay platforms (ELISAs), the U.S. SARS-CoV-2 standard is measured on the same 96-well plate as the daily assay standard, run as serial dilutions in triplicate and quadruplicate (Fig. 1). Standard curves are constructed for both the U.S. SARS-CoV-2 Serology standard and daily assay standard. A test of parallelism and linearity between the two dose-response curves is then performed to ensure that immunoaffinity differences or matrix effects do not prevent accurate calibration with the U.S. SARS-CoV-2 serology standard. Units based on the U.S. SARS-CoV-2 serology standard can then be assigned to the assay daily standard, to harmonize assays and units for result reporting. For non-plate-based assay platforms, similar dilution-based standard curves are constructed.

FIG 1.

FIG 1

Open in a new tab

Example plate map for assay calibration setup. Numbers indicate suggested serial dilutions. Serial dilutions of primary and secondary calibrators (reference materials) are plated in triplicate, and the daily internal assay standard is plated in quadruplicate. C_STD, daily internal assay standard; STD-C1, -C2, and -C3, primary calibrator (primary reference material or standard); STD-T1, -T2, and T3, secondary calibrator (secondary reference material or standard); NEG, negative control sample; PC1, positive control sample 1; PC2, positive control sample 2.

Traceability of the FNLCR standard to the first WHO international standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136) was established, to allow SeroNet assays to convert U.S. serology standard units to WHO IS units. WHO IS 20/136 is a freeze-dried equivalent of 0.25 mL of pooled plasma from 11 individuals with a history of SARS-CoV-2 infection. Once reconstituted, the WHO standard has an arbitrary unitage of 1,000 binding antibody units (BAU)/mL. Eight serial dilutions of the U.S. SARS-CoV-2 serology standard and WHO IS 20/136 were run in triplicate. Parallel line analysis, which included tests for parallelism and linearity, was utilized to assign WHO IS 20/136 standard units to the U.S. SARS-CoV-2 serology standard; this will allow SeroNet institutions to convert U.S. SARS-CoV-2 serology standard units to WHO standard units for serological methods.

Patient consent statement.

This work involves a descriptive summary of serological assays and assay harmonization plans and does not include factors necessitating patient consent.

ACKNOWLEDGMENTS

This work was funded by NCI contract no. 75N91019D00024, task order no. 75N91021F00001, award numbers 21X089 (J.L., V.M., J.P., J.Q., and L.S.), 21X090 (J.M.C. and N.C.O.), 21X091 (A.B.K., S.N.T., and B.T.), and 21X092 (C.C.-C., A.F.-B., F.K., D.R.M., V.S., and A.W.) and NCI grants U54CA260591 (K.S.), U01CA260469 (T.L., D.A.G., S.W.G., C.D.H., K.K., and N.P.), U54CA260543 (L.P.), U54CA260582 (S.-L.L. and G.L.), U54CA260492 (S.L.K. and S.D.), U54CA260563 (F.E.-H.L., M.S.S., N.S.H., J.L.D., and J.D.R.), U01CA260541 (J.D.B., A.K.P., T.L.S., E.T.S., C.A.S., P.P., and A.M.E.), U01CA260526 (K.W.B., J.C.F., and J.L.K.), U01CA261276 (R.A.B., C.F., and A.M.M.), U01CA260539 (C.L.K.), U01CA260508 (L.M.S., A.P.D., R.C.G., D.T.H., W.T.L., J.L.Y., and A.F.P.), and U01CA260462 (S.B. and S.P.).

A.B.K. is a consultant for Roche Diagnostics and has received research support from Siemens Healthcare Diagnostics and Kyowa Kirin Pharmaceutical Development. J.D.B., A.K.P., E.T.S., and T.L.S. have received research support from Altimmune. J.D.R. and M.S.S. are coinventors on a patent filed by Emory University covering the serology assay described in this paper. M.S.S. serves on the advisory board for Moderna and Ocugen. F.E.-H.L. is the founder of MicroB-plex, Inc. J.L.D. is the CSO of MicroB-plex, Inc. N.S.H. has been a senior scientist at MicroB-plex, Inc. F.E.-H.L. has research grants from the Gates Foundation and Genentech, is on the Scientific Advisory Board (SAB) of Be Biopharma, Inc., and received royalties from BLI, Inc., as an inventor for the plasma cell survival media. S.B. has research support from Merck and Pfizer, and is a member of the CMV Vaccine Advisory Committees of Merck and Moderna. S.P. has research support from Moderna. D.A.G. is the chief scientific and strategy advisor of Salimetrics, LLC. S.W.G. is the chief scientific officer of Salimetrics, LLC. Mount Sinai has licensed serological assays to commercial entities and has filed for patent protection for serological assays. The Icahn School of Medicine at Mount Sinai has filed patent applications relating to the COVID-19 serological assay (“Serology Assay”) and NDV-based SARS-CoV-2 vaccines which list F.K. (“Serology Assay,” vaccines), V.S. (“Serology Assay”), A.F-B. (“Serology Assay”), D.R.M. (“Serology Assay”), and C.C.-C. (“Serology Assay”) as coinventors. The foundational “Serology Assay” intellectual property (IP) was licensed by the Icahn School of Medicine at Mount Sinai to commercial entities, including Kantaro Biosciences, a company in which Mount Sinai has a financial interest. F.K. has consulted for Merck and Pfizer (before 2020) and is currently consulting for Pfizer, Third Rock Ventures, Seqirus, and Avimex. F.K.’s laboratory is also collaborating with Pfizer on animal models of SARS-CoV-2. All remaining authors report no relevant conflicts of interest.

Contributor Information

Amy B. Karger, Email: karge026@umn.edu.

Michael J. Imperiale, University of Michigan—Ann Arbor

REFERENCES

  • 1.National Cancer Institute. 2022. NCI Serological Sciences Network for COVID-19 (SeroNet). https://www.cancer.gov/research/key-initiatives/covid-19/coronavirus-research-initiatives/serological-sciences-network. Accessed 2 December 2021.
  • 2.Food and Drug Administration (FDA). In vitro diagnostics EUAs—serology and other adaptive immune response tests for SARS-CoV-2. https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-authorizations-medical-devices/in-vitro-diagnostics-euas-serology-and-other-adaptive-immune-response-tests-sars-cov-2. Accessed 2 December 2021.
  • 3.Muecksch F, Wise H, Batchelor B, Squires M, Semple E, Richardson C, McGuire J, Clearly S, Furrie E, Greig N, Hay G, Templeton K, Lorenzi JCC, Hatziioannou T, Jenks S, Bieniasz PD. 2021. Longitudinal serological analysis and neutralizing antibody levels in coronavirus disease 2019 convalescent patients. J Infect Dis 223:389–398. doi: 10.1093/infdis/jiaa659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Perez-Saez J, Zaballa M-E, Yerly S, Andrey DO, Meyer B, Eckerle I, Balavoine J-F, Chappuis F, Pittet D, Trono D, Kherad O, Vuilleumier N, Kaiser L, Guessous I, Stringhini S, Azman AS, Specchio-COVID19 Study Group . 2021. Persistence of anti-SARS-CoV-2 antibodies: immunoassay heterogeneity and implications for serosurveillance. Clin Microbiol Infect 27:1695.e7–e1695.e12. doi: 10.1016/j.cmi.2021.06.040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Muecksch F, Wise H, Templeton K, Batchelor B, Squires M, McCance K, Jarvis L, Malloy K, Furrie E, Richardson C, MacGuire J, Godber I, Burns A, Mavin S, Zhang F, Schmidt F, Bieniasz P, Jenks S, Hatziioannou T. 2021. Longitudinal variation in SARS-CoV-2 antibody levels and emergence of viral variants: implications for the ability of serological assays to predict immunity. medRxiv doi: 10.1101/2021.07.02.21259939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Narowski TM, Raphel K, Adams LE, Huang J, Vielot NA, Jadi R, de Silva AM, Baric RS, Lafleur JE, Premkumar L. 2022. SARS-CoV-2 mRNA vaccine induces robust specific and cross-reactive IgG and unequal neutralizing antibodies in naive and previously infected people. Cell Rep 38:110336. doi: 10.1016/j.celrep.2022.110336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Sholukh AM, Fiore-Gartland A, Ford ES, Miner MD, Hou YJ, Tse LV, Kaiser H, Zhu H, Lu J, Madarampalli B, Park A, Lempp FA, St Germain R, Bossard EL, Kee JJ, Diem K, Stuart AB, Rupert PB, Brock C, Buerger M, Doll MK, Randhawa AK, Stamatatos L, Strong RK, McLaughlin C, Huang M-L, Jerome KR, Baric RS, Montefiori D, Corey L. 2021. Evaluation of cell-based and surrogate SARS-CoV-2 neutralization assays. J Clin Microbiol 59:e00527-21. doi: 10.1128/JCM.00527-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.von Rhein C, Scholz T, Henss L, Kronstein-Wiedemann R, Schwarz T, Rodionov RN, Corman VM, Tonn T, Schnierle BS. 2021. Comparison of potency assays to assess SARS-CoV-2 neutralizing antibody capacity in COVID-19 convalescent plasma. J Virol Methods 288:114031. doi: 10.1016/j.jviromet.2020.114031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Olbrich L, Castelletti N, Schälte Y, Garí M, Pütz P, Bakuli A, Pritsch M, Kroidl I, Saathoff E, Guggenbuehl Noller JM, Fingerle V, Le Gleut R, Gilberg L, Brand I, Falk P, Markgraf A, Deák F, Riess F, Diefenbach M, Eser T, Weinauer F, Martin S, Quenzel EM, Becker M, Durner J, Girl P, Müller K, Radon K, Fuchs C, Wölfel R, Hasenauer J, Hoelscher M, Wieser A, on behalf of the KoCo-Study Group . 2021. Head-to-head evaluation of seven different seroassays including direct viral neutralisation in a representative cohort for SARS-CoV-2. J Gen Virol 102:001653. doi: 10.1099/jgv.0.001653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Röltgen K, Powell AE, Wirz OF, Stevens BA, Hogan CA, Najeeb J, Hunter M, Wang H, Sahoo MK, Huang C, Yamamoto F, Manohar M, Manalac J, Otrelo-Cardoso AR, Pham TD, Rustagi A, Rogers AJ, Shah NH, Blish CA, Cochran JR, Jardetzky TS, Zehnder JL, Wang TT, Narasimhan B, Gombar S, Tibshirani R, Nadeau KC, Kim PS, Pinsky BA, Boyd SD. 2020. Defining the features and duration of antibody responses to SARS-CoV-2 infection associated with disease severity and outcome. Sci Immunol 5:54. doi: 10.1126/sciimmunol.abe0240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Alter G, Yu J, Liu J, Chandrashekar A, Borducchi EN, Tostanoski LH, McMahan K, Jacob-Dolan C, Martinez DR, Chang A, Anioke T, Lifton M, Nkolola J, Stephenson KE, Atyeo C, Shin S, Fields P, Kaplan I, Robins H, Amanat F, Krammer F, Baric RS, Le Gars M, Sadoff J, de Groot AM, Heerwegh D, Struyf F, Douoguih M, van Hoof J, Schuitemaker H, Barouch DH. 2021. Immunogenicity of Ad26.COV2.S vaccine against SARS-CoV-2 variants in humans. Nature 596:268–272. doi: 10.1038/s41586-021-03681-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Amanat F, Stadlbauer D, Strohmeier S, Nguyen THO, Chromikova V, McMahon M, Jiang K, Asthagiri Arunkumar G, Jurczyszak D, Polanco J, Bermudez-Gonzalez M, Kleiner G, Aydillo T, Miorin L, Fierer D, Amarilis Lugo L, Milunka Kojic E, Stoever J, Liu STH, Cunningham-Rundles C, Felgner PL, Moran T, Garcia-Sastre A, Caplivski D, Cheng A, Kedzierska K, Vapalahti O, Hepojoki JM, Simon V, Krammer F. 2020. A serological assay to detect SARS-CoV-2 seroconversion in humans. medRxiv doi: 10.1101/2020.03.17.20037713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Arthur JM, Forrest JC, Boehme KW, Kennedy JL, Owens S, Herzog C, Liu J, Harville TO. 2021. Development of ACE2 autoantibodies after SARS-CoV-2 infection. PLoS One 16:e0257016. doi: 10.1371/journal.pone.0257016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Collier A-RY, McMahan K, Yu J, Tostanoski LH, Aguayo R, Ansel J, Chandrashekar A, Patel S, Apraku Bondzie E, Sellers D, Barrett J, Sanborn O, Wan H, Chang A, Anioke T, Nkolola J, Bradshaw C, Jacob-Dolan C, Feldman J, Gebre M, Borducchi EN, Liu J, Schmidt AG, Suscovich T, Linde C, Alter G, Hacker MR, Barouch DH. 2021. Immunogenicity of COVID-19 mRNA vaccines in pregnant and lactating women. JAMA 325:2370–2380. doi: 10.1001/jama.2021.7563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Klein SL, Pekosz A, Park H-S, Ursin RL, Shapiro JR, Benner SE, Littlefield K, Kumar S, Naik HM, Betenbaugh MJ, Shrestha R, Wu AA, Hughes RM, Burgess I, Caturegli P, Laeyendecker O, Quinn TC, Sullivan D, Shoham S, Redd AD, Bloch EM, Casadevall A, Tobian AA. 2020. Sex, age, and hospitalization drive antibody responses in a COVID-19 convalescent plasma donor population. J Clin Invest 130:6141–6150. doi: 10.1172/JCI142004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Premkumar L, Segovia-Chumbez B, Jadi R, Martinez DR, Raut R, Markmann A, Cornaby C, Bartelt L, Weiss S, Park Y, Edwards CE, Weimer E, Scherer EM, Rouphael N, Edupuganti S, Weiskopf D, Tse LV, Hou YJ, Margolis D, Sette A, Collins MH, Schmitz J, Baric RS, de Silva AM. 2020. The receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients. Sci Immunol 5:eabc8413. doi: 10.1126/sciimmunol.abc8413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Salazar E, Kuchipudi SV, Christensen PA, Eagar T, Yi X, Zhao P, Jin Z, Long SW, Olsen RJ, Chen J, Castillo B, Leveque C, Towers D, Lavinder J, Gollihar J, Cardona J, Ippolito G, Nissly R, Bird I, Greenawalt D, Rossi RM, Gontu A, Srinivasan S, Poojary I, Cattadori IM, Hudson PJ, Josleyn NM, Prugar L, Huie K, Herbert A, Bernard DW, Dye JM, Kapur V, Musser JM. 2020. Convalescent plasma anti-SARS-CoV-2 spike protein ectodomain and receptor-binding domain IgG correlate with virus neutralization. J Clin Invest 130:6728–6738. doi: 10.1172/JCI141206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Seegmiller JC, Kokaisel EL, Story SJ, Zaun CP, Peters JM, Thomas SN, Karger AB. 2020. Method comparison of SARS-CoV-2 serology assays involving three commercially available platforms and a novel in-house developed enzyme-linked immunosorbent assay. Clin Biochem 86:34–35. doi: 10.1016/j.clinbiochem.2020.08.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Stadlbauer D, Amanat F, Chromikova V, Jiang K, Strohmeier S, Arunkumar GA, Tan J, Bhavsar D, Capuano C, Kirkpatrick E, Meade P, Brito RN, Teo C, McMahon M, Simon V, Krammer F. 2020. SARS-CoV-2 seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup. Curr Protoc Microbiol 57:e100. doi: 10.1002/cpmc.100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Stadlbauer D, Tan J, Jiang K, Hernandez MM, Fabre S, Amanat F, Teo C, Arunkumar GA, McMahon M, Capuano C, Twyman K, Jhang J, Nowak MD, Simon V, Sordillo EM, van Bakel H, Krammer F. 2021. Repeated cross-sectional sero-monitoring of SARS-CoV-2 in New York City. Nature 590:146–150. doi: 10.1038/s41586-020-2912-6. [DOI] [PubMed] [Google Scholar]
  • 21.Suthar MS, Zimmerman MG, Kauffman RC, Mantus G, Linderman SL, Hudson WH, Vanderheiden A, Nyhoff L, Davis CW, Adekunle O, Affer M, Sherman M, Reynolds S, Verkerke HP, Alter DN, Guarner J, Bryksin J, Horwath MC, Arthur CM, Saakadze N, Smith GH, Edupuganti S, Scherer EM, Hellmeister K, Cheng A, Morales JA, Neish AS, Stowell SR, Frank F, Ortlund E, Anderson EJ, Menachery VD, Rouphael N, Mehta AK, Stephens DS, Ahmed R, Roback JD, Wrammert J. 2020. Rapid generation of neutralizing antibody responses in COVID-19 patients. Cell Rep Med 1:100040. doi: 10.1016/j.xcrm.2020.100040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Thomas SN, Altawallbeh G, Zaun CP, Pape KA, Peters JM, Titcombe PJ, Dileepan T, Rapp MJ, Bold TD, Schacker TW, Arbefeville S, Ferrieri P, Thyagarajan B, Jenkins MK, Karger AB. 2021. Initial determination of COVID-19 seroprevalence among outpatients and healthcare workers in Minnesota using a novel SARS-CoV-2 total antibody ELISA. Clin Biochem 90:15–22. doi: 10.1016/j.clinbiochem.2021.01.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Vidal SJ, Collier A-RY, Yu J, McMahan K, Tostanoski LH, Ventura JD, Aid M, Peter L, Jacob-Dolan C, Anioke T, Chang A, Wan H, Aguayo R, Ngo D, Gerszten RE, Seaman MS, Barouch DH. 2021. Correlates of neutralization against SARS-CoV-2 variants of concern by early pandemic sera. J Virol 95:e00404-21. doi: 10.1128/JVI.00404-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Zeng C, Evans JP, Pearson R, Qu P, Zheng Y-M, Robinson RT, Hall-Stoodley L, Yount J, Pannu S, Mallampalli RK, Saif L, Oltz E, Lozanski G, Liu S-L. 2020. Neutralizing antibody against SARS-CoV-2 spike in COVID-19 patients, health care workers, and convalescent plasma donors. JCI Insight 5:e143213. doi: 10.1172/jci.insight.143213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Sariol C, Pantoja P, Serrano-Collazo C, Rosa-Arocho T, Armina-Rodríguez A, Cruz L, Stone E, Arana T, Climent C, Latoni G, Atehortua D, Pabon-Carrero C, Pinto A, Brien J, Espino A. 2021. Function is more reliable than quantity to follow up the humoral response to the receptor-binding domain of SARS-CoV-2-spike protein after natural infection or COVID-19 vaccination. Viruses 13:1972. doi: 10.3390/v13101972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Roy V, Fischinger S, Atyeo C, Slein M, Loos C, Balazs A, Luedemann C, Astudillo MG, Yang D, Wesemann DR, Charles R, Lafrate AJ, Feldman J, Hauser B, Caradonna T, Miller TE, Murali MR, Baden L, Nilles E, Ryan E, Lauffenburger D, Beltran WG, Alter G. 2020. SARS-CoV-2-specific ELISA development. J Immunol Methods 484–485:112832. doi: 10.1016/j.jim.2020.112832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Rosenberg ES, Tesoriero JM, Rosenthal EM, Chung R, Barranco MA, Styer LM, Parker MM, John Leung S-Y, Morne JE, Greene D, Holtgrave DR, Hoefer D, Kumar J, Udo T, Hutton B, Zucker HA. 2020. Cumulative incidence and diagnosis of SARS-CoV-2 infection in New York. Ann Epidemiol 48:23–29.e4. doi: 10.1016/j.annepidem.2020.06.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Styer LM, Hoen R, Rock J, Yauney E, Nemeth K, Bievenue R, Parker MM. 2021. High-throughput multiplex SARS-CoV-2 IgG microsphere immunoassay for dried blood spots: a public health strategy for enhanced serosurvey capacity. Microbiol Spectr 9:e00134-21. doi: 10.1128/Spectrum.00134-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Lee WT, Girardin RC, Dupuis AP, Kulas KE, Payne AF, Wong SJ, Arinsburg S, Nguyen FT, Mendu DR, Firpo-Betancourt A, Jhang J, Wajnberg A, Krammer F, Cordon-Cardo C, Amler S, Montecalvo M, Hutton B, Taylor J, McDonough KA. 2021. Neutralizing antibody responses in COVID-19 convalescent sera. J Infect Dis 223:47–55. doi: 10.1093/infdis/jiaa673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Yates JL, Ehrbar DJ, Hunt DT, Girardin RC, Dupuis AP, Payne AF, Sowizral M, Varney S, Kulas KE, Demarest VL, Howard KM, Carson K, Hales M, Ejemel M, Li Q, Wang Y, Peredo-Wende R, Ramani A, Singh G, Strle K, Mantis NJ, McDonough KA, Lee WT. 2021. Serological analysis reveals an imbalanced IgG subclass composition associated with COVID-19 disease severity. Cell Rep Med 2:100329. doi: 10.1016/j.xcrm.2021.100329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Pisanic N, Randad PR, Kruczynski K, Manabe YC, Thomas DL, Pekosz A, Klein SL, Betenbaugh MJ, Clarke WA, Laeyendecker O, Caturegli PP, Larman HB, Detrick B, Fairley JK, Sherman AC, Rouphael N, Edupuganti S, Granger DA, Granger SW, Collins MH, Heaney CD. 2020. COVID-19 serology at population scale: SARS-CoV-2-specific antibody responses in saliva. J Clin Microbiol 59:e02204-20. doi: 10.1128/JCM.02204-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Canaday DH, Carias L, Oyebanji OA, Keresztesy D, Wilk D, Payne M, Aung H, St Denis K, Lam EC, Wilson B, Rowley CF, Berry SD, Cameron CM, Cameron MJ, Balazs AB, Gravenstein S, King CL. 2021. Reduced BNT162b2 mRNA vaccine response in SARS-CoV-2-naive nursing home residents. Clin Infect Dis 73:2112–2115. doi: 10.1093/cid/ciab447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Tian L, Elsheikh EB, Patrone PN, Kearsley AJ, Gaigalas AK, Inwood S, Lin-Gibson S, Esposito D, Wang L. 2021. Towards quantitative and standardized serological and neutralization assays for COVID-19. Int J Mol Sci 22:2723. doi: 10.3390/ijms22052723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Su Y, Chen D, Yuan D, Lausted C, Choi J, Dai CL, Voillet V, Duvvuri VR, Scherler K, Troisch P, Baloni P, Qin G, Smith B, Kornilov SA, Rostomily C, Xu A, Li J, Dong S, Rothchild A, Zhou J, Murray K, Edmark R, Hong S, Heath JE, Earls J, Zhang R, Xie J, Li S, Roper R, Jones L, Zhou Y, Rowen L, Liu R, Mackay S, O’Mahony DS, Dale CR, Wallick JA, Algren HA, Zager MA, Wei W, Price ND, Huang S, Subramanian N, Wang K, Magis AT, Hadlock JJ, Hood L, Aderem A, Bluestone JA, Lanier LL, ISB-Swedish COVID19 Biobanking Unit, et al. 2020. Multi-omics resolves a sharp disease-state shift between mild and moderate COVID-19. Cell 183:1479–1495.e20. doi: 10.1016/j.cell.2020.10.037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Xhangolli I, Dura B, Lee G, Kim D, Xiao Y, Fan R. 2019. Single-cell analysis of CAR-T cell activation reveals a mixed TH1/TH2 response independent of differentiation. Genomics Proteomics Bioinformatics 17:129–139. doi: 10.1016/j.gpb.2019.03.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Heaney CD, Pisanic N, Randad PR, Kruczynski K, Howard T, Zhu X, Littlefield K, Patel EU, Shrestha R, Laeyendecker O, Shoham S, Sullivan D, Gebo K, Hanley D, Redd AD, Quinn TC, Casadevall A, Zenilman JM, Pekosz A, Bloch EM, Tobian AAR. 2021. Comparative performance of multiplex salivary and commercially available serologic assays to detect SARS-CoV-2 IgG and neutralization titers. J Clin Virol 145:104997. doi: 10.1016/j.jcv.2021.104997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Haddad NS, Nguyen DC, Kuruvilla ME, Morrison-Porter A, Anam F, Cashman KS, Ramonell RP, Kyu S, Saini AS, Cabrera-Mora M, Derrico A, Alter D, Roback JD, Horwath M, O'Keefe JB, Wu HM, Wong A-KI, Dretler AW, Gripaldo R, Lane AN, Wu H, Chu HY, Lee S, Hernandez M, Engineer V, Varghese J, Patel R, Jalal A, French V, Guysenov I, Lane CE, Mengistsu T, Normile KE, Mnzava O, Le S, Sanz I, Daiss JL, Lee FE-H. 2021. One-stop serum assay identifies COVID-19 disease severity and vaccination responses. Immunohorizons 5:322–335. doi: 10.4049/immunohorizons.2100011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Amanat F, White KM, Miorin L, Strohmeier S, McMahon M, Meade P, Liu W-C, Albrecht RA, Simon V, Martinez-Sobrido L, Moran T, García-Sastre A, Krammer F. 2020. An in vitro microneutralization assay for SARS-CoV-2 serology and drug screening. Curr Protoc Microbiol 58:e108. doi: 10.1002/cpmc.108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Wajnberg A, Amanat F, Firpo A, Altman DR, Bailey MJ, Mansour M, McMahon M, Meade P, Mendu DR, Muellers K, Stadlbauer D, Stone K, Strohmeier S, Simon V, Aberg J, Reich DL, Krammer F, Cordon-Cardo C. 2020. Robust neutralizing antibodies to SARS-CoV-2 infection persist for months. Science 370:1227–1230. doi: 10.1126/science.abd7728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Hassert M, Geerling E, Stone ET, Steffen TL, Feldman MS, Dickson AL, Class J, Richner JM, Brien JD, Pinto AK. 2020. mRNA induced expression of human angiotensin-converting enzyme 2 in mice for the study of the adaptive immune response to severe acute respiratory syndrome coronavirus 2. PLoS Pathog 16:e1009163. doi: 10.1371/journal.ppat.1009163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Vanderheiden A, Edara VV, Floyd K, Kauffman RC, Mantus G, Anderson E, Rouphael N, Edupuganti S, Shi P-Y, Menachery VD, Wrammert J, Suthar MS. 2020. Development of a rapid focus reduction neutralization test assay for measuring SARS-CoV-2 neutralizing antibodies. Curr Protoc Immunol 131:e116. doi: 10.1002/cpim.116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Girardin RC, Dupuis AP, Payne AF, Sullivan TJ, Strauss D, Parker MM, McDonough KA. 2021. Temporal analysis of serial donations reveals decrease in neutralizing capacity and justifies revised qualifying criteria for coronavirus disease 2019 convalescent plasma. J Infect Dis 223:743–751. doi: 10.1093/infdis/jiaa803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Dhakal S, Ruiz-Bedoya CA, Zhou R, Creisher PS, Villano JS, Littlefield K, Ruelas Castillo J, Marinho P, Jedlicka AE, Ordonez AA, Bahr M, Majewska N, Betenbaugh MJ, Flavahan K, Mueller ARL, Looney MM, Quijada D, Mota F, Beck SE, Brockhurst J, Braxton AM, Castell N, Stover M, D’Alessio FR, Metcalf Pate KA, Karakousis PC, Mankowski JL, Pekosz A, Jain SK, Klein SL. 2021. Sex differences in lung imaging and SARS-CoV-2 antibody responses in a COVID-19 golden Syrian hamster model. mBio 12:e00974-21. doi: 10.1128/mBio.00974-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Morgenlander WR, Henson SN, Monaco DR, Chen A, Littlefield K, Bloch EM, Fujimura E, Ruczinski I, Crowley AR, Natarajan H, Butler SE, Weiner JA, Li MZ, Bonny TS, Benner SE, Balagopal A, Sullivan D, Shoham S, Quinn TC, Eshleman SH, Casadevall A, Redd AD, Laeyendecker O, Ackerman ME, Pekosz A, Elledge SJ, Robinson M, Tobian AA, Larman HB. 2021. Antibody responses to endemic coronaviruses modulate COVID-19 convalescent plasma functionality. J Clin Invest 131:e146927. doi: 10.1172/JCI146927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Ogega CO, Skinner NE, Blair PW, Park HS, Littlefield K, Ganesan A, Dhakal S, Ladiwala P, Antar AA, Ray SC, Betenbaugh MJ, Pekosz A, Klein SL, Manabe YC, Cox AL, Bailey JR. 2021. Durable SARS-CoV-2 B cell immunity after mild or severe disease. J Clin Invest 131:e145516. doi: 10.1172/JCI145516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Kared H, Redd AD, Bloch EM, Bonny TS, Sumatoh H, Kairi F, Carbajo D, Abel B, Newell EW, Bettinotti MP, Benner SE, Patel EU, Littlefield K, Laeyendecker O, Shoham S, Sullivan D, Casadevall A, Pekosz A, Nardin A, Fehlings M, Tobian AA, Quinn TC. 2021. SARS-CoV-2-specific CD8+ T cell responses in convalescent COVID-19 individuals. J Clin Invest 131:e145476. doi: 10.1172/JCI145476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Patel EU, Bloch EM, Clarke W, Hsieh Y-H, Boon D, Eby Y, Fernandez RE, Baker OR, Keruly M, Kirby CS, Klock E, Littlefield K, Miller J, Schmidt HA, Sullivan P, Piwowar-Manning E, Shrestha R, Redd AD, Rothman RE, Sullivan D, Shoham S, Casadevall A, Quinn TC, Pekosz A, Tobian AAR, Laeyendecker O. 2021. Comparative performance of five commercially available serologic assays to detect antibodies to SARS-CoV-2 and identify individuals with high neutralizing titers. J Clin Microbiol 59:e02257-20. doi: 10.1128/JCM.02257-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Röltgen K, Nielsen SCA, Arunachalam PS, Yang F, Hoh RA, Wirz OF, Lee AS, Gao F, Mallajosyula V, Li C, Haraguchi E, Shoura MJ, Wilbur JL, Wohlstadter JN, Davis MM, Pinsky BA, Sigal GB, Pulendran B, Nadeau KC, Boyd SD. 2021. mRNA vaccination compared to infection elicits an IgG-predominant response with greater SARS-CoV-2 specificity and similar decrease in variant spike recognition. medRxiv doi: 10.1101/2021.04.05.21254952. [DOI] [Google Scholar]
  • 49.Pegu A, O’Connell S, Schmidt SD, O’Dell S, Talana CA, Lai L, Albert J, Anderson E, Bennett H, Corbett KS, Flach B, Jackson L, Leav B, Ledgerwood JE, Luke CJ, Makowski M, Roberts PC, Roederer M, Rebolledo PA, Rostad CA, Rouphael NG, Shi W, Wang L, Widge AT, Yang ES, mRNA-1273 Study Group, Beigel JH, Graham BS, Mascola JR, Suthar MS, McDermott A, Doria-Rose NA. 2021. Durability of mRNA-1273-induced antibodies against SARS-CoV-2 variants. bioRxiv doi: 10.1101/2021.05.13.444010. [DOI] [PMC free article] [PubMed]
  • 50.Weissman D, Alameh M-G, de Silva T, Collini P, Hornsby H, Brown R, LaBranche CC, Edwards RJ, Sutherland L, Santra S, Mansouri K, Gobeil S, McDanal C, Pardi N, Hengartner N, Lin PJC, Tam Y, Shaw PA, Lewis MG, Boesler C, Şahin U, Acharya P, Haynes BF, Korber B, Montefiori DC. 2021. D614G spike mutation increases SARS CoV-2 susceptibility to neutralization. Cell Host Microbe 29:23–31.e4. doi: 10.1016/j.chom.2020.11.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Figueiredo JC, Hirsch FR, Kushi LH, Nembhard WN, Crawford JM, Mantis N, Finster L, Merin NM, Merchant A, Reckamp KL, Melmed GY, Braun J, McGovern D, Parekh S, Corley DA, Zohoori N, Amick BC, Du R, Gregersen PK, Diamond B, Taioli E, Sariol C, Espino A, Weiskopf D, Gifoni A, Brien J, Hanege W, Lipsitch M, Zidar DA, McAlearney AS, Wajnberg A, LaBaer J, Lewis EY, Binder RA, Moormann AM, Forconi C, Forrester S, Batista J, Schieffelin J, Kim D, Biancon G, VanOudenhove J, Halene S, Fan R, Barouch DH, Alter G, Pinninti S, Boppana SB, Pati SK, Latting M, et al. 27 April 2022. Mission, organization and future direction of the Serological Sciences Network for COVID-19 (SeroNet) Epidemiologic Cohort Studies. Open Forum Infect Dis doi: 10.1093/ofid/ofac171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.WHO. 2022. WHO manual for the preparation of reference materials for use as secondary standards in antibody testing. https://www.who.int/publications/m/item/who-manual-for-reference-material-for-antibody-testing. Accessed 20 May 2022.
  • 53.Espino AM, Pantoja P, Sariol CA. 2020. Validation and performance of a quantitative IgG assay for the screening of SARS-CoV-2 antibodies. bioRxiv 2020.06.11.146332. doi: 10.1101/2020.06.11.146332. [DOI]
  • 54.Boehme KW, Kennedy JL, Snowden J, Owens SM, Kouassi M, Mann RL, Paredes A, Putt C, James L, Jin J, Du R, Kirkpatrick C, Modi Z, Caid K, Zohoori N, Kothari A, Boyanton BL, Jr, Forrest JC. 2021. Pediatric SARS-CoV-2 seroprevalence in Arkansas over the first year of the COVID-19 pandemic. medRxiv 2021.08.04.21261592. doi: 10.1101/2021.08.04.21261592. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from mSphere are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES