NSAE 2020 [A].

Study characteristics
Patient Sampling	Purpose: Detection of prior infection with SARS‐CoV‐22 Design: Two‐group study to derive sensitivity and specificity [1] Samples from SARS‐CoV‐2 RT‐PCR‐positive participants including patients admitted to hospital or identified through surveillance of HCWs (n = 158) >= 18 years old at Oxford University Hospital NHS Foundation Trust and volunteer plasma donors (n = 378) (total n = 536) via NHS Blood and Transplant, across UK [2] Pre‐pandemic BioBank samples (n = 976) Recruitment: Not reported but appeared consecutive based on reporting of sample inclusion and PRISMA flow diagram Prospective or retrospective: Retrospective Sample size: 1512 (536) Further detail: All samples from individuals > 18 years old. (Four sources of known positives documented: Gastrointestinal illness sub‐study, ISARIC/WHO Clinical Characterisation Protocol for Severe Emerging Infections, Sepsis Immunomics project, and volunteer plasma donors) Inclusion: [1] Healthcare workers and patients >= 18 years old or plasma donors >= 18 years old with a previous positive SARS‐CoV‐2 RT‐PCR nose/throat swab, with blood samples taken ≥ 20 days post‐symptom onset [2] Healthy individuals 30‐50 years old, collected between 2015‐2018 Exclusion: Laboratory labelling mix up: n = 7; date of PCR or symptom onset not recorded: n = 3; samples missing: n = 2; insufficient sample to evaluate across all 4 platforms: n = 5 [1] De‐duplication by individual: n = 96; < 20 days post‐symptom onset/PCR‐positive test: n = 126
Patient characteristics and setting	Setting: Mixed Location: Oxford University Hospital NHS Foundation Trust, Oxfordshire, UK Country: United Kingdom Dates: 1/2/20‐31/5/20 Symptoms and severity: [1a] Varied severity at the time of sampling; asymptomatic n = 13, mild n = 122, severe n = 16 and critical/death n = 7 [1b] All convalescent Demographics: Not reported, aged > 18 years Exposure history: Not stated Non‐Covid group 1: Pre‐pandemic samples Source: Oxford BioBank ‐ samples collected between Sept 2014 and Oct 2016 Characteristics: Healthy individuals aged 30‐50 years old Non‐Covid group 2: NA
Index tests	Test name: [A]‐ SARS‐CoV‐2 IgG assay [B]‐ LIAISON SARS‐CoV‐2 S1/S2 IgG assay [C]‐ Elecsys Anti‐SARS‐CoV‐2 assay [D]‐ SARS‐CoV‐2 Total assay [Additional in‐house assay evaluated 'Oxford immunoassay'; not eligible for this review] Manufacturer: [A]‐ Abbott, Chicago, IL, USA [B]‐ DiaSorin, Saluggia, Italy [C]‐ Roche, Basel, Switzerland [D]‐ Siemens, Munich, Germany Antibody: A & B‐ IgG C & D‐ Total Ab Antigen target: A & C‐ Nucleocapsid B‐ Spike‐Protein S1/S2 D‐ Spike‐Protein S1 RBD Evaluation setting: Laboratory Test method: Not Stated Timing of samples: Of 158 admitted patients and HCWs median 36.5 d pso (IQR 28, 53; range 20 to 73) All 378 volunteer plasma donors were ≥ 28 days pso Samples used: Serum or plasma Test operator: Trained laboratory staff in UK Accreditation Service (UKAS) accredited laboratories: [A] [C] John Radcliffe Hospital Clinical Biochemistry and Microbiologylaboratories in Oxford [B] [D] PHE Porton Down Definition of test positivity: Table S2 appendix [A]‐ Positive: ≥ 1.4 [B]‐ Positive: ≥ 15.0 AU/mL [C]‐ Reactive: ≥ 1.0 [D]‐ Reactive: ≥ 1.0 Blinding reported: Yes Threshold predefined: Yes, as per manufacturers' instructions; alternative thresholds also explored, e.g. to optimise specificity
Target condition and reference standard(s)	Reference standard: RT‐PCR; assays used not described Samples used: "Nose or throat swab" Timing of reference standard: Not stated Blinded to index test: Yes, on basis of timing and COVID‐19 group recruited on basis of positive RT‐PCR Incorporated index test: No. Definition of non‐COVID cases: Pre‐pandemic Samples used: NA Timing of reference standard: Pre‐pandemic controls Blinded to index test: Yes, as based on pre‐pandemic controls Incorporated index test: No
Flow and timing	Time interval between index and reference tests: [1a] Median 27 (range 3‐59) days (n = 105) [1b] Median 44 (range 32‐82) days All patients received same reference standard: No Missing data: None; stated 'No samples failed testing on any of the four commercial platforms.' Uninterpretable results: None reported Indeterminate results: Equivocal results reported separately; considered as index negative for purposes of this review Unit of analysis: Patients; stated "samples were de‐duplicated by individual, and the latest sample from each individual was analysed".
Comparative
Notes	Funding: Public Health England and UK National Institute for Health Research This work was supported by the UK National Institute for Health Research (NIHR) Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance at the University of Oxford, Oxford, UK in partnership with Public Health England, and the NIHR Oxford Biomedical Research Centre. Publication status: Published Paper Source: Lancet Infectious Diseases 2020 Author COI: None RC reported personal fees and reported acting as a co‐founder and consultant at MIROBIO, a University of Oxford spinout. The company targets immune inhibitory receptors as treatments for inflammation and auto‐immune disease. This work is unrelated to the serology work. DWE has received lecture fees from Gilead, outside of the submitted work. MGS reported grants from the UK Department of Health and Social Care, National Institute of Health Research UK, Medical Research Council UK, Health Protection Research Unit in Emerging and Zoonotic Infections, University of Liverpool, Liverpool, UK, during the conduct of the study; and acting as a member of the Infectious Disease Scientific Advisory Board to Integrum Scientific, Greensboro, NC, USA, outside of the submitted work. All other authors declared no competing interests.
Methodological quality
Item	Authors' judgement	Risk of bias	Applicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?	Yes
Was a case‐control design avoided?	No
Did the study avoid inappropriate exclusions?	Unclear
Did the study avoid inappropriate inclusions?	Yes
Could the selection of patients have introduced bias?		High risk
Are there concerns that the included patients and setting do not match the review question?			High
DOMAIN 2: Index Test (All tests)
DOMAIN 2: Index Test (Antibody tests)
Were the index test results interpreted without knowledge of the results of the reference standard?	Yes
If a threshold was used, was it pre‐specified?	Yes
Could the conduct or interpretation of the index test have introduced bias?		Low risk
Are there concerns that the index test, its conduct, or interpretation differ from the review question?			Low concern
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?	Yes
Were the reference standard results interpreted without knowledge of the results of the index tests?	Yes
The reference standard does not incorporate the index test	Yes
Could the reference standard, its conduct, or its interpretation have introduced bias?		Low risk
Are there concerns that the target condition as defined by the reference standard does not match the question?			High
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?	Unclear
Did all patients receive the same reference standard?	No
Were all patients included in the analysis?	Yes
Did all participants receive a reference standard?	Yes
Were results presented per patient?	Yes
Could the patient flow have introduced bias?		High risk