3d919f81f731568f6c3e0adbb51742f6 VIR_a2025v17n716.pdf 20b2be8252d3aabfc5e54b01c58f30f84b169d75 VIR_a2025v17n716.pdf f4afb4af457edde43c65f076636b6d9f94ee8366de33afc3c3a64e285e8fed55 VIR_a2025v17n716.pdf Title: Comparison of Three Commercial ELISA Kits for Detection of Antibodies Against SARS-CoV-2 in Serum Samples from Different Animal Species Subject: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the coronavirus disease 19 (COVID-19) pandemic, significantly impacting global health, economies, and social stability. In February 2020, the first cases of SARS-CoV-2 infections in animals were documented, highlighting the potential risks posed by regular human–animal interactions in facilitating viral transmission. In consequence, it is essential to validate surveillance methods for SARS-CoV-2 in animals. In the present study, 101 sera from different animal species (36 cats, 41 dogs, 4 ferrets, 10 wild boar, 6 domestic goats, and 4 lions) were tested using three different ELISA kits to evaluate humoral responses against SARS-CoV-2. ELISA results were compared and correlated with a pseudovirus neutralization test (pVNT), considered as the reference assay. ELISA-1, targeting the receptor binding domain (RBD) neutralizing antibodies (nAbs) of SARS-CoV-2, exhibited the highest diagnostic performance, and proved to be a reliable tool for initial screenings in high-throughput animal studies. In contrast, ELISA-2 (also targeting RBD nAbs) and ELISA-3 (targeting nucleoprotein antibodies) demonstrated lower sensitivity for detecting seropositive animals. Keywords: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); humoral immune response; animal serology; ELISA; pseudovirus neutralization test (pVNT); one health; zoonosis Author: Leira Fernández-Bastit, Sílvia Marfil, Edwards Pradenas, Julià Blanco, Júlia Vergara-Alert and Joaquim Segalés Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Fri May 16 12:49:15 2025 CEST ModDate: Mon May 19 07:52:10 2025 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 16 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1642164 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- WNWTFR+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 FNSQWZ+VnURWPalladioL Type 1 Custom yes yes yes 16 0 JSKUOV+URWPalladioL-Roma Type 1 Custom yes yes yes 21 0 VRGGUE+URWPalladioL-Bold Type 1 Custom yes yes yes 27 0 EEVECG+URWPalladioL-Ital Type 1 Custom yes yes yes 32 0 IALKNK+CMSY10 Type 1 Builtin yes yes yes 61 0 EMMNCG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 79 0 EMMNCH+PalatinoLinotype CID TrueType Identity-H yes yes yes 82 0 EMMNCI+PalatinoLinotype TrueType WinAnsi yes yes no 88 0 EMMNCK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 91 0 EMMNHK+PalatinoLinotype TrueType MacRoman yes yes no 94 0 EMMNCG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 108 0 EMMNCH+PalatinoLinotype CID TrueType Identity-H yes yes yes 111 0 EMMNCI+PalatinoLinotype TrueType WinAnsi yes yes no 117 0 EMMNCK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 120 0 EMMNHK+PalatinoLinotype TrueType MacRoman yes yes no 123 0 EMMNCG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 140 0 EMMNCH+PalatinoLinotype CID TrueType Identity-H yes yes yes 143 0 EMMNCI+PalatinoLinotype TrueType WinAnsi yes yes no 149 0 EMMNCK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 152 0 EMMNHK+PalatinoLinotype TrueType MacRoman yes yes no 155 0 EMMNCG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 167 0 EMMNCH+PalatinoLinotype CID TrueType Identity-H yes yes yes 170 0 EMMNCI+PalatinoLinotype TrueType WinAnsi yes yes no 176 0 EMMNCK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 179 0 EMMNHK+PalatinoLinotype TrueType MacRoman yes yes no 182 0 EMMNCG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 197 0 EMMNCH+PalatinoLinotype CID TrueType Identity-H yes yes yes 200 0 EMMNCI+PalatinoLinotype TrueType WinAnsi yes yes no 206 0 EMMNCK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 209 0 EMMNHK+PalatinoLinotype TrueType MacRoman yes yes no 212 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-09-19 03:48:14 CEST RepresentationInformation: VIR_a2025v17n716.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-09-18 14:11:02 CEST Size: 1642164 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 465 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Study Design Destination: subsection.2.1 Item: Title: Antibody Detection Tests Destination: subsection.2.2 Children: Item: Title: ELISA-1: cPass SARS-CoV-2 Neutralization Antibody Detection Kit Destination: subsubsection.2.2.1 Item: Title: ELISA-2: SARS-CoV-2 NeutraLISA Kit Destination: subsubsection.2.2.2 Item: Title: ELISA-3: ID Screen® SARS-CoV-2 Double Antigen Multi-Species Assay Destination: subsubsection.2.2.3 Item: Title: Pseudovirus Neutralization Assay (pVNT) Destination: subsection.2.3 Item: Title: Statistical Analyses Destination: subsection.2.4 Item: Title: Results Destination: section.3 Children: Item: Title: Detection of SARS-CoV-2 Humoral Response by pVNT Destination: subsection.3.1 Item: Title: Detection of SARS-CoV-2 Humoral Response by ELISA-1, ELISA-2, and ELISA-3 Destination: subsection.3.2 Item: Title: Qualitative Comparison of ELISAs with pVNT Destination: subsection.3.3 Item: Title: Correlation of ELISAs Detecting RBD nAbs (ELISA-1 vs. ELISA-2) Destination: subsection.3.4 Item: Title: Qualitative and Quantitative Correlation of RBD nAbs (ELISA-1) and N Protein Antibodies (ELISA-3) Destination: subsection.3.5 Item: Title: Serological Assays Associated with RT-qPCR Detection in Animal Samples Destination: subsection.3.6 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: appendix.A. Info: Title: Comparison of Three Commercial ELISA Kits for Detection of Antibodies Against SARS-CoV-2 in Serum Samples from Different Animal Species Author: Leira Fernández-Bastit, Sílvia Marfil, Edwards Pradenas, Julià Blanco, Júlia Vergara-Alert and Joaquim Segalés Subject: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the coronavirus disease 19 (COVID-19) pandemic, significantly impacting global health, economies, and social stability. In February 2020, the first cases of SARS-CoV-2 infections in animals were documented, highlighting the potential risks posed by regular human animal interactions in facilitating viral transmission. In consequence, it is essential to validate surveillance methods for SARS-CoV-2 in animals. In the present study, 101 sera from different animal species (36 cats, 41 dogs, 4 ferrets, 10 wild boar, 6 domestic goats, and 4 lions) were tested using three different ELISA kits to evaluate humoral responses against SARS-CoV-2. ELISA results were compared and correlated with a pseudovirus neutralization test (pVNT), considered as the reference assay. ELISA-1, targeting the receptor binding domain (RBD) neutralizing antibodies (nAbs) of SARS-CoV-2, exhibited the highest diagnostic performance, and proved to be a reliable tool for initial screenings in high-throughput animal studies. In contrast, ELISA-2 (also targeting RBD nAbs) and ELISA-3 (targeting nucleoprotein antibodies) demonstrated lower sensitivity for detecting seropositive animals. 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