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The objective of this study was to develop a bead-based Multiplexed Fluorometric ImmunoAssay (MFIA) as an improved serological surveillance method compared to the<jats:italic>Salmonella<\/jats:italic>mix ELISA, which has been the national reference immunoassay in the Danish<jats:italic>Salmonella<\/jats:italic>surveillance program for about 20\u00a0years.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>An MFIA for detection of antibodies to<jats:italic>Salmonella<\/jats:italic>serogroup B and C<jats:sub>1<\/jats:sub>was developed and optimized with regard to coupling of beads with<jats:italic>Salmonella<\/jats:italic>lipopolysaccharide antigens and establishing suitable assay conditions. The<jats:italic>Salmonella<\/jats:italic>MFIA was validated by testing sera from experimentally infected pigs as well as field sera from non-infected and infected pig herds, and by comparing to results from the<jats:italic>Salmonella<\/jats:italic>mix ELISA, which was run in parallel. Sensitivity and specificity was evaluated using receiver operating curve analysis showing an area under curve for the serogroup B and C<jats:sub>1<\/jats:sub>MFIA of 0.984 and 0.998, respectively. The<jats:italic>Salmonella<\/jats:italic>MFIA was shown to detect more antibody-positive samples in seropositive herds compared to the<jats:italic>Salmonella<\/jats:italic>mix ELISA, and Bayesian statistics confirmed that the MFIA had a considerably higher sensitivity (94.5%) compared to the mix ELISA (75.1%). The assay specificity was slightly lower for the<jats:italic>Salmonella<\/jats:italic>MFIA (96.8%) compared to<jats:italic>Salmonella<\/jats:italic>mix ELISA (99.5%). Coupled beads were stable for at least 1\u00a0year at 4\u02daC, and MFIA reproducibility and repeatability of the<jats:italic>Salmonella<\/jats:italic>MFIA were acceptable. Results from proficiency tests also indicated that the<jats:italic>Salmonella<\/jats:italic>MFIA was more sensitive than the<jats:italic>Salmonella<\/jats:italic>mix ELISA and that they had similar specificity.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>A bead-based MFIA for simultaneous detection of porcine serum antibodies to<jats:italic>Salmonella enterica<\/jats:italic>serogroup B and C<jats:sub>1<\/jats:sub>was developed and implemented in the Danish porcine serological<jats:italic>Salmonella<\/jats:italic>surveillance program in 2018. The<jats:italic>Salmonella<\/jats:italic>MFIA can distinguish, as opposed to the<jats:italic>Salmonella<\/jats:italic>mix ELISA, between antibodies to serogroup B and C<jats:sub>1<\/jats:sub>and the MFIA shows considerably better sensitivity.<\/jats:p><\/jats:sec>","DOI":"10.1186\/s12917-022-03362-w","type":"journal-article","created":{"date-parts":[[2022,7,5]],"date-time":"2022-07-05T07:02:54Z","timestamp":1657004574000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Development and evaluation of a bead-based Multiplexed Fluorescent ImmunoAssay (MFIA) for detection of antibodies to Salmonella enterica serogroup B and C1 in pigs"],"prefix":"10.1186","volume":"18","author":[{"given":"Sanne Schou","family":"Berger","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ulla","family":"Riber","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vibeke Fr\u00f8kj\u00e6r","family":"Jensen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Klara T\u00f8lb\u00f8ll","family":"Lauritsen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lars Ole","family":"Andresen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2022,7,5]]},"reference":[{"key":"3362_CR1","doi-asserted-by":"crossref","unstructured":"Griffith RW, Carlson SA, Krull AC. 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These animal experiments had been performed in accordance with guidelines and regulations and had been approved by the Danish Animal Experiments Inspectorate.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare no competing interests or other interests that might be perceived to influence the results and\/or discussion reported in this paper.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"259"}}