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The current panzootic with H5 clade 2.3.4.4b has caused numerous, widespread outbreaks in various domestic and wild avian species with high mortalities, massive losses, and a high frequency of spillover events to unexpected novel mammalian hosts, such as dairy cows. The global H5N1 situation raises serious concerns about zoonotic risks due to effective mammal-to-mammal transmission. Therefore, it is critical to increase surveillance intensity of a broadened species range, particularly at the human-animal interface. For this purpose, reliable and cost-effective serological tools that are easy to perform and suitable for high-throughput screening are critically needed. The newly developed double-antigen enzyme-linked immunosorbent assay format employing a luminescence-based detection technology has demonstrated compliance with such prerequisites. The assay allowed sensitive and specific detection of antibodies directed against H5 hemagglutinin of clade 2.3.4.4b in a wide range of birds and mammals, including humans. Furthermore, it allowed differentiating H5 anti-head-specific from cross-reacting anti-stalk antibodies, which represents a valuable feature with regard to the monitoring of future vaccination programs with H5-specific vaccines. Thus, the assay is a significant contribution to existing serological diagnostic tests for a clade-optimized and species-independent detection of influenza A virus antibodies.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>The ongoing highly pathogenic avian influenza virus H5N1 panzootic has caused numerous outbreaks in domestic and wild animals, with frequent spillover events to unexpected host species, which underscores the importance of intensified surveillance. However, sensitive and specific multi-species serological assays represent a major gap. For this purpose, we developed a novel double-antigen enzyme-linked immunosorbent assay that employs an innovative luminescence-based readout strategy. The test allowed a highly sensitive and specific detection of H5-specific antibodies in a wide range of avian and mammalian species, including humans. It therefore represents a valuable contribution to improving species-independent serological diagnostic tools for the detection of influenza A virus antibodies.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/jcm.01907-25","type":"journal-article","created":{"date-parts":[[2026,8,13]],"date-time":"2026-08-13T18:38:29Z","timestamp":1786646309000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Development of a multi-species luciferase-based double-antigen ELISA for the detection of antibodies against influenza A virus H5 clade 2.3.4.4b"],"prefix":"10.1128","volume":"64","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-9274-1895","authenticated-orcid":false,"given":"Andrea","family":"Aebischer","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/04xv01a59","id-type":"ROR","asserted-by":"publisher"}],"name":"Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Federal 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