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The genus Staphylococcus contains many pathogenic strains that are difficult to differentiate. Given the absence of a specific immunological test to identify autochthonous species, we have characterized soluble antigens (SAgs) using hyperimmune sera from BABL/c mice. Ten samples were taken from the farmers’ hands and cattle udders on three different farms. The isolated species were identified using the API kit (Staph) and their ability to form biofilms was determined. The species most commonly found in the isolates (90%) corresponded to the coagulase-negative bacteria and Staphylococcus sciuri (S. sciuri), which presented the ability of biofilm formation, representing the majority (60%) in this group. We produced SAgs from those Staphylococcus species present in a higher frequency, such as S. sciuri, S. aureus, and the reference strain, S. aureus ATCC 6835. Polyclonal antibodies (PAb) from mice allowed SAgs characterization by enzyme-linked immunoassay (ELISA) and immunoblotting. The humoral response obtained with the PAb by indirect ELISA tests indicated that our hyperimmune sera have a high recognition for all SAgs produced. We also evaluated the hyperimmune sera cross-reactivity between different SAgs by indirect ELISA and immunoblotting assays. The ELISA experiments showed a significant statistical difference in the recognition of S. sciuri when compared to SAgs from S. aureus. These results showed a high antigenicity and specificity from S. sciuri SAgs in immune tests. We identified a specific immunodominant polypeptide of ~31 kDa (p31) from S. sciuri SAg, which do not presented cross-reactivity between different SAgs. We concluded that the p31 polypeptide from S. sciuri SAg could be used as antigen in a differential diagnosis test for different staphylococcal species. Full article