Jung 2020a.

Study characteristics
Patient Sampling	Purpose: Diagnosis of acute‐phase infection Design: Multi‐group study to establish sensitivity and specificity. [1] Non‐COVID cases (57 samples) [1a] Non‐COVID cases with negative rt‐PCR test for SARS‐CoV‐2 (38 samples) for "clinical specificity", [1b] Non‐COVID cases with other diseases (cross‐reaction panel, 19 samples) for "analytical specificity". [2] confirmed COVID‐19 patients (n = 104 samples) for "clinical sensitivity". [2a] Only 42 inpatients for "seroconversion" study had days pso. Of these, only 19 samples had an eligible time split for our review. Recruitment: Not specified Prospective or retrospective: prospective (serum/plasma was not frozen but stored for up to 5 days at 4 °C until analysis) Sample size: 161 (104) samples of which 76 (19) samples were eligible for our review Further detail: Inclusion ‐ [2] COVID‐19 confirmed by PCR, [2a] Patients who were repeatedly assessed in our hospital, positive for SARS‐CoV‐2 by RT‐PCR, and had a known date of symptom onset [1a] negative for SARS‐CoV‐2 by RT‐PCR; [1b] patients with other confirmed viral infections; negative for SARS‐CoV‐2 by RT‐PCR or no known exposure, travel history, or symptoms of COVID‐19 No exclusion criteria defined
Patient characteristics and setting	Setting: [2] Not specified [2a] Hospital inpatients Location: [2] Texas Children’s Hospital or other in the Texas Medical Center (Baylor St. Luke’s and Ben Taub Hospitals), Houston, Texas [2a] Texas Children’s Hospital, Houston, Texas Country: Texas, USA Dates: Not specified (before 2020 August) Symptoms and severity: Not specified Demographics: Not specified Exposure history: Not stated Non‐Covid group 1: [1a] rt‐PCR‐negative samples (healthy volunteers?) Source: Not specified Characteristics: Not specified (negative SARS CoV‐2 RT‐PCR results) Non‐Covid group 2: [2b] Cross‐reaction panel Source: Concurrent, not stated Characteristics: Samples known to be positive for other viruses by molecular testing (including Influenza A, Influenza B, respiratory syncytial virus (RSV), adenovirus, rhinovirus), but negative for SARS‐CoV‐2 by RT‐PCR (3 samples did not have RT‐PCR result, but had no known exposure, travel history, or symptoms of COVID‐19)
Index tests	Test name: Ash Laboratories SARS‐CoV2 IgG and IgM ELISA Immunoassay Manufacturer: Ash Laboratories Antibody: IgG and IgM Antigen target: nucleocapsid (N) and spike (S) proteins. Evaluation setting: Laboratory Test method: ELISA (on Dynex‐DS2 automated immunoassay system) Timing of samples: [2a] < 6 days pso: n = 10, 6‐14 days pso: n = 9, > 14 days pso: n = 24 for [A] and n = 22 for [B]. Samples used: peripheral venous blood; plasma or serum stored for up to 5 days at 4 °C until analysis. Test operator: Not stated (different operators for [1a] and [2]) Definition of test positivity: > 12 AU/mL reactive; < 10 AU/mL non‐reactive, 10–12 AU/mL equivocal Blinding reported: Not stated Threshold predefined: Yes
Target condition and reference standard(s)	Reference standard: [2] COVID‐19 by RT‐PCR or TMA (Transcription‐mediated amplification) Samples used: Not stated Timing of reference standard: Not stated Blinded to index test: Yes (based on timing of tests) Incorporated index test: No Definition of non‐COVID cases: contemporaneous [1a] negative SARS CoV‐2 RT‐PCR results [1b] negative SARS CoV‐2 RT‐PCR results; 3 samples no known exposure, travel history, or symptoms of COVID‐19 Samples used: Not stated Timing of reference standard: Not stated Blinded to index test: Yes (based on timing of tests) Incorporated index test: No
Flow and timing	Time interval between index and reference tests: Not stated All patients received same reference standard: no (rt‐PCR and TMA) [1] rt‐PCR [2a] rt‐PCR Remaining of [2] rt‐PCR and TMA Missing data: Yes, number of samples with IgM results lower than for IgG results (see Tables 2 and 3) Uninterpretable results: Not stated Indeterminate results: yes, but equivocal samples were considered positive Unit of analysis: [1a] Not stated [1b] Patients [2a] Samples
Comparative
Notes	Funding: EG and JJ were supported by the Ching Nan Ou Fellowship Endowment. Some of the validation kits used in this study were provided by Ansh Laboratories, but they did not participate in study design, validation, or data interpretation. Publication status: Published paper Source: Clinical Chimica Acta 510 (2020) 790–5 Author COI: Some of the validation kits used in this study were provided by Ansh Laboratories, but they did not participate in study design, validation, or data interpretation.
Methodological quality
Item	Authors' judgement	Risk of bias	Applicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?	Unclear
Was a case‐control design avoided?	No
Did the study avoid inappropriate exclusions?	Unclear
Did the study avoid inappropriate inclusions?	No
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?	Unclear
If a threshold was used, was it pre‐specified?	Yes
Could the conduct or interpretation of the index test have introduced bias?		Unclear 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?	No
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?		High 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?	Yes
Were all patients included in the analysis?	No
Did all participants receive a reference standard?	No
Were results presented per patient?	Unclear
Could the patient flow have introduced bias?		High risk