af171fb811c41175845c4baa870dd294 VETSC_a2025v12p995.pdf 808548d938314395623f0377bbf1325dce28710a VETSC_a2025v12p995.pdf a0ed658bb5a9dad8333453810524d4dfc53b70240ac793b709540cb271dfd1a1 VETSC_a2025v12p995.pdf Title: Development and Standardization of Indirect ELISA for African Swine Fever Virus Using Recombinant p30 Protein Produced in Prokaryotic System Subject: African Swine Fever (ASF), caused by the African Swine Fever Virus (ASFV), is a highly contagious hemorrhagic disease with high mortality (100%) in pigs and is considered the most devastating disease to date. Given the importance of this disease, we aimed to assess the use of the recombinant p30 protein as the sole antigen for the development of an accurate and precise ELISA test (iELISA) for the virus. The recombinant p30 protein (rp30) was produced in a bacterial expression system using a SUMO-tagged expression vector. Protein expression was confirmed by Western blot analysis and purified using affinity chromatography. Antigenicity was evaluated in CF-1 mice, which demonstrated the ability to generate high levels of specific antibodies. The rp30 showed a sensitivity of 95.6% when used in the development of iELISA, a specificity of 92.3%, and a kappa index () of 0.836. Furthermore, reference sera (OIE-ASF) were used to validate the assays, and the results demonstrated an excellent capacity to detect ASF antibodies using only the rp30 antigen up to a serum dilution of 1:100. The inter- and intra-assay variability coefficients were 4.27% and 4.85%, respectively, demonstrating that the assay was accurate and reproducible, allowing its use in seroepidemiological analyses for ASF surveillance. Keywords: p30; African swine fever; synthetic gene; recombinant protein; serovigilance Author: José Luis Cerriteño-Sánchez, José Bryan García-Cambrón, Perla Lucero Zavala-Ocampo, Llilianne Ganges and Julieta Sandra Cuevas-Romero Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Thu Oct 16 08:18:20 2025 CEST ModDate: Thu Oct 16 08:24:51 2025 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2039957 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- EYQFQP+VnURWPalladioL Type 1 Custom yes yes yes 106 0 EMTAET+URWPalladioL-Bold Type 1 Custom yes yes yes 107 0 MCWVDJ+URWPalladioL-Roma Type 1 Custom yes yes yes 108 0 MDCIFP+CMSY10 Type 1 Builtin yes yes yes 109 0 OYXAVF+URWPalladioL-Ital Type 1 Custom yes yes yes 110 0 ZSBMBZ+EURM10 Type 1 Builtin yes yes yes 111 0 GIGFZE+CMR10 Type 1 Builtin yes yes yes 206 0 VKNCQV+CMEX10 Type 1 Builtin yes yes yes 207 0 HJIHLO+PalatinoLinotype TrueType WinAnsi yes yes no 240 0 HJIHLO+PalatinoLinotype TrueType WinAnsi yes yes no 327 0 HJIHLO+PalatinoLinotype TrueType WinAnsi yes yes no 280 0 HJIHLO+PalatinoLinotype TrueType WinAnsi yes yes no 367 0 HJIHLO+PalatinoLinotype TrueType WinAnsi yes yes no 353 0 HJIHLO+PalatinoLinotype TrueType WinAnsi yes yes no 375 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-11-21 04:24:31 CET RepresentationInformation: VETSC_a2025v12p995.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-11-20 14:29:36 CET Size: 2039957 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 527 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Materials and Methods Children: Item: Title: Cloning and Overexpression of ASFV-p30 Protein Item: Title: Antigenic Evaluation of ASFV-rp30 Protein In Vivo Item: Title: Optimization of ASFV-rp30 Coated Indirect ELISA Item: Title: Statistical Analysis Item: Title: Indirect ELISA Cut-Off Determination Item: Title: Results Children: Item: Title: Development and Expression of the Recombinant System for rp30 Item: Title: Humoral Evaluation of rp30 Item: Title: Standardization and Validation of the iELISA Using rp30 as the Antigen Item: Title: Cut-Off Point, Sensitivity, Specificity, and Kappa Index of the iELISA Item: Title: Discussion Item: Title: Conclusions Item: Title: References Info: Title: Development and Standardization of Indirect ELISA for African Swine Fever Virus Using Recombinant p30 Protein Produced in Prokaryotic System Author: José Luis Cerriteño-Sánchez, José Bryan García-Cambrón, Perla Lucero Zavala-Ocampo, Llilianne Ganges and Julieta Sandra Cuevas-Romero Subject: African Swine Fever (ASF), caused by the African Swine Fever Virus (ASFV), is a highly contagious hemorrhagic disease with high mortality (100%) in pigs and is considered the most devastating disease to date. Given the importance of this disease, we aimed to assess the use of the recombinant p30 protein as the sole antigen for the development of an accurate and precise ELISA test (iELISA) for the virus. The recombinant p30 protein (rp30) was produced in a bacterial expression system using a SUMO-tagged expression vector. Protein expression was confirmed by Western blot analysis and purified using affinity chromatography. Antigenicity was evaluated in CF-1 mice, which demonstrated the ability to generate high levels of specific antibodies. The rp30 showed a sensitivity of 95.6% when used in the development of iELISA, a specificity of 92.3%, and a kappa index () of 0.836. Furthermore, reference sera (OIE-ASF) were used to validate the assays, and the results demonstrated an excellent capacity to detect ASF antibodies using only the rp30 antigen up to a serum dilution of 1:100. The inter- and intra-assay variability coefficients were 4.27% and 4.85%, respectively, demonstrating that the assay was accurate and reproducible, allowing its use in seroepidemiological analyses for ASF surveillance. 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