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

Aebischer, A.; Gunther, A.; Piesche, R.; Wernike, K.; Murr, M.; Harder, T. C.; Hoffmann, D.; Grund, C.; Bellido Martin, B.; Fouchier, R. A. M.; Kreer, C.; Daniel, K.; Ullrich, L.; Sprute, R.; Roller, M.; Reese, L.; Wassermann, J.; Schares, G.; Kuryshko, M.; Abdelwhab, E.; Beer, M.

doi:10.64898/2026.01.05.697617

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bioRxiv · 10.64898/2026.01.05.697617

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

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Abstract

The highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 represent a major threat to animal and public health. 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 screenings are critically needed. The newly developed double antigen ELISA format employing a luminescence-based detection technology has demonstrated to comply with such prerequisites. The assay allowed the detection of H5-specific antibodies even early after infection or vaccination in a wide range of birds and mammals including humans. It further demonstrated superior analytical sensitivity and high specificity for antibodies directed against H5 hemagglutinin of clade 2.3.4.4b as no cross-reactivity with other hemagglutinin subtypes was observed. Thus, the assay represents a valuable contribution to existing serological diagnostic tests for a clade-optimized detection of influenza A virus antibodies in a broad range of species. ImportanceThe ongoing HPAIV H5N1 panzootic has caused numerous outbreaks in domestic and wild animals with frequent spillover events to unexpected host species, which underscores the importance of an intensified surveillance. However, sensitive and specific multi-species serological assays represent a major gap. For this purpose, we developed a novel double antigen ELISA which employs an innovative luminescence-based read-out strategy. The test allowed a highly sensitive and specific detection of H5-specific antibodies even early after infection or vaccination in a wide range of avian and mammalian species including humans. It therefore represents a significant contribution to improving species-independent serological diagnostic tools for the detection of influenza A virus antibodies.

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Aebischer, A., Gunther, A., Piesche, R., Wernike, K., Murr, M., Harder, T. C., Hoffmann, D., Grund, C., Bellido Martin, B., Fouchier, R. A. M., Kreer, C., Daniel, K., Ullrich, L., Sprute, R., Roller, M., Reese, L., Wassermann, J., Schares, G., Kuryshko, M., Abdelwhab, E., Beer, M.. 2026-01-05. 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. https://doi.org/10.64898/2026.01.05.697617

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