Evaluation of serologic assays for detection and quantification of influenza antibodies in milk samples from H5N1 highly pathogenic avian influenza virus infected dairy cows

Since the confirmation of H5N1 HPAI virus in US dairy cattle on March 25, 2024, the bovine HPAI outbreak has continued with more than 1,000 confirmed herds in 19 states reported as of April 2, 2026. To ensure ongoing monitoring of HPAI spread, additional serologic assays have been evaluated to complement existing commercial ELISAs and molecular tools that underpin current epidemiological approaches. We previously established an N1 antibody-specific neuraminidase inhibition (NI)-enzyme linked lectin assay (ELLA) for DIVA (differentiation of infected from vaccinated animals) surveillance to detect H5N1 infection in vaccinated poultry. In this study, we applied NI-ELLA for the detection and quantification of N1 antibody in individual (n = 150) and bulk tank (n = 104) bovine milk samples. We demonstrated that both untreated samples and infranatant collected by centrifugation followed by heat inactivation can be used for different serologic assays depending on sample quality. There was a strong correlation in antibody detection between NI-ELLA and commercial ELISAs. Endpoint titers determined by NI-ELLA also correlated well with virus neutralization titers, which are used to evaluate protective antibody levels in milk samples. Detection and quantification of N1 antibodies in bulk tank milk samples further showed higher detection rates and stronger antibody titers among virus isolation-negative samples (regardless of the presence or absence of viral RNA detected by PCR) compared to virus isolation-positive samples. Higher levels of neutralizing antibody likely contributed to the lack of infectious virus detection depending on the stage of viral infection when milk was collected. Future studies are warranted to describe the duration and dynamics of antibody responses in milk samples over extended periods across individual cows, and NI-ELLA will serve as a valuable quantitative tool for evaluating antibody responses in those studies.