The optimization of a rapid dot-blot immunoassay for the detection of Salmonella enteritidis

Abstract

The aim of this study was to optimize the Enzyme Linked ImmunoSorbent Assay (ELISA) for detection of Salmonella enteritidis. Commercially available media were compared with whole egg homogenate for relative ability to resuscitate and propagate S. enteritidis to detectable levels. Incubation in whole egg homogenate, tripticase soy broth (TSB), and lactose broth (LB) resulted in comparable numbers of S. enteritidis. Results of the immunoassay found that TSB gave the strongest visual representation showing a positive test for S. enteritidis. Incubation time necessary to detect one S. enteriridis colony forming unit was reduced from 20 to 10 h using TSB as the enrichment broth. Addition of detergent before incubation had a negligible negative effect on the growth of this organism. When S. enteritidis was incubated with a mixed flora of (1:10$\sp2$ CFU/mL) competitive microorganisms, in either TSB or in homogenized whole egg supplemented with ferrous sulphate it was able to reproduce to detectable numbers for the immunoassay. When environmental samples were tested, the same ratio of S. enteritidis to competitive flora was detected. The antibody was purified and freeze-dried in the presence of various exipients and its stability over time was tested by ELISA. The antibody was stable over the storage period of 70 days when stored alone and with trehalose or mannitol as cryoprotectant, even at storage conditions of 50$\sp\circ$C. Maltose and sucrose degraded the antibody at elevated temperatures. This is thought to be due to participation in Maillard browning reaction between the reducing sugars and the antibody. (Abstract shortened by UMI.)