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As both ASF and CSF are notifiable diseases under the World Organisation for Animal Health (WOAH), rapid and accurate identification is crucial for effective outbreak management. In this study, we developed a multicolor lateral flow immunoassay (LFIA) based on latex microspheres (LMs) for the simultaneous detection of antibodies against ASF virus (ASFV) and CSF virus (CSFV). The assay enables visual differentiation within 15\u2009min, with red indicating ASFV antibodies and blue indicating CSFV antibodies. After optimization, the LFIA demonstrated a sensitivity of 1:256, equivalent to that of a commercial ASFV ELISA kit and four\u2010fold higher than that for CSFV (1:64). The assay exhibited high specificity, showing no cross\u2010reactivity with other common swine pathogens and bovine viral diarrhea virus (BVDV). When applied to 180 clinical serum samples and compared with commercial ELISA kits, the LFIA achieved Cohen\u2019s kappa values of 0.986 for ASFV and 0.918 for CSFV, indicating excellent agreement. Additionally, intra and interbatch evaluations confirmed its robust repeatability. Overall, the multicolor LM\u2010LFIA offers a rapid, sensitive, specific, and cost\u2010effective tool for point\u2010of\u2010care testing (POCT) of ASFV and CSFV antibodies, holding promise for routine field surveillance and disease control.<\/jats:p>","DOI":"10.1155\/tbed\/5512419","type":"journal-article","created":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T09:29:35Z","timestamp":1768555775000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Establishment and Evaluation of a Multicolor Latex Microsphere\u2010Based Lateral Flow Immunoassay for the Simultaneous Detection of Antibodies Against African and Classical Swine Fever 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