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J Med Virol . Fluorescent Microsphere Immunoassay for Isotype-Specific H5N1 Antibody Detection in Serum and Milk Samples From Dairy Cattle: A Tool f

tetano

Editor, Senior Moderator
J Med Virol


. 2025 Apr;97(4):e70321.
doi: 10.1002/jmv.70321. Fluorescent Microsphere Immunoassay for Isotype-Specific H5N1 Antibody Detection in Serum and Milk Samples From Dairy Cattle: A Tool for Epidemiological Surveillance

Maritza Cordero-Ortiz[SUP] 1 [/SUP], Ronaldo Magtoto[SUP] 2 [/SUP], Brooklyn Cauwels[SUP] 2 [/SUP], David H Baum[SUP] 2 [/SUP], Bailey Arruda[SUP] 3 [/SUP], Patrick J Gorden[SUP] 2 [/SUP], Drew R Magstadt[SUP] 2 [/SUP], Jesús Hernández[SUP] 1 [/SUP], Luis G Giménez-Lirola[SUP] 2 [/SUP]



Affiliations
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.

Keywords: FMIA; HPAI H5N1; Luminex; dairy cattle; epidemiological surveillance; fluorescent microsphere immunoassay; influenza A virus.

 
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