A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed using monoclonal antibodies (MAbs) as a rapid, economical alternative to culture isolation procedures for detection of Salmonella. Four MAbs previously shown to react with Salmonella strains representing 18 different serogroups were evaluated as capture antibodies and, after biotinylation, as detection antibodies. One MAb (M183) was selected for use in the ELISA to capture and detect Salmonella antigens. The detection limit of the ELISA was evaluated using Salmonella enterica subspecies enterica serovar Typhimurium and various selective and nonselective Salmonella enrichment media. The highest detection limit (ca. 10(4) CFU/ml) was achieved using an enrichment broth containing brain heart infusion, yeast extract, sodium hydrogen selenite, and sodium cholate (BYSC) after preenrichment in buffered peptone water. The ELISA detected all Salmonella serovars tested, which included representative serovars of serogroups B, C, D, and E and gave negative results for all non-Salmonella species tested. Samples (106) from various sources, including fecal samples from humans and pigeons, chicken carcass rinses, chicken parts, feed, and the environment, were used to evaluate the performance of the ELISA. The ELISA had a specificity and sensitivity of 100 and 91%, respectively, and a kappa value of 0.93 relative to the culture methods. Such an ELISA has the potential to be used in the implementation of the pathogen reduction and hazard analysis critical control point systems as well as in clinical laboratories.
An enzyme-linked i m j l t r m * o n assay (ELLFA} and a microtitre plate entymelinked immunosorbent msay (ELLSA) were developed and compared for their ability to detect staphylococcal enterotoxin B (SEB). Ihe double antibody capture format was used for both assays. Factors which improved the sensitivity of the ELIFA system were (1) addition of casein and thimerosal to the antigen dilution buffer;(2) addition of polyethylene glycol (MW 6OOO) to the detection and conjugate antibody dilution buffers: and (3) washing with diethanolamine bufer prior to addition of the substrate/chromgen. Ihe ELIFA system had a turnaround time of approximately 1 h and a detection limit of 1 ng/mL of purified SEB. n e ELISA had a total turnaround time of 21 h, or 3 h using plates pre-coated overnight with the capture antibody. Ihe detection limit of the EUSA for purijied SEB was 0.05 ng/mL. ?he detection limit of SEB in cheese samples spiked with puriped enterotoxin and subjected to a simple extraction procedure was 1 nglmL and 0.1 ng/mL of extract, with the ELIFA and the ELISA, respectively. lCorrespondmg author. Tel. (613) 998-9320 ext. 5924, Fax (613) 954-5876, E-Mail Address: valdivia@ern.agr.ca
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