Detection and Isolation of Yersinia pestis Without Fraction 1 Antigen by Monoclonal Antibody in Foods and Water

Abstract

Most available immunoassays for Yersinia pestis are based on the detection of fraction 1 antigen (F1) when yersiniae are grown at 37°C. A monoclonal antibody (MAb) was developed based on the detection of surface antigens that are not F1. F1-deficient Y. pestis cells were induced and used to immunize BALB/c mice from which MAb (immunoglobulin G1), which specifically recognizes Y. pestis, with or without F1, was obtained. This MAb (6B5) did not cross-react with enteric bacteria, including Yersinia enterocolitica. Enzyme-linked immunosorbent assay results revealed that MAb 6B5 is specific for Y. pestis, with the exception of a minor cross-reaction with Yersinia pseudotuberculosis. Western immunoblot analysis revealed that MAb 6B5 recognizes a Y. pestis outer membrane protein of ca. 30 kDa. Magnetic beads that were coated with MAb 6B5 were compared with beads coated with polyclonal antibody (PAb; rabbit) against Y. pestis for the isolation of Y. pestis in food and water samples by using a PATHATRIX cell concentration apparatus. Enrichment cultures of Y. pestis in different foods by using two different times (6 and 24 h) in brain heart infusion broth at 37°C were evaluated. Results revealed MAb 6B5–coated magnetic beads were equivalent to magnetic beads coated with PAb against Y. pestis A1122 whole cells in concentrating Y. pestis for isolation, especially when samples were enriched for 6 h. However, the selectivity for Y. pestis of the magnetic beads coated with MAb 6B5 was greater than that coated with PAb.