Abstract
The detection of highly pathogenic avian influenza A virus H5N1 in dairy cows in the United States underscores the urgent need for reliable laboratory tools to support epidemiological surveillance. This study describes the development and evaluation of a fluorescent microsphere immunoassay (FMIA) for detecting IgG, IgA, and IgM antibodies against the H5 hemagglutinin protein in serum and milk samples from dairy cattle, with results compared to the NP-ELISA. H5/FMIA demonstrated 100% diagnostic sensitivity and 99.7% diagnostic specificity for anti-H5 IgG antibodies in serum. In milk samples, the assay showed comparable performance for IgG and IgA, achieving 94.7% sensitivity and 98% specificity. Paired serum and milk samples exhibited stronger correlations using H5/FMIA (r = 0.88 for IgG, r = 0.82 for IgA, and r = 0.58 for IgM) than NP-ELISA (r = 0.54). H5/FMIA IgA showed greater sensitivity in "early" infections, whereas IgG was more robust in "late" cases. These findings confirm the utility of H5/FMIA as a valuable antibody isotype-specific tool for serodiagnosis and epidemiological surveillance of H5N1 in dairy cattle.