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Mansfield, L.P., 1999. The rapid detection of Salmonella by an IMS-ELISA system. PhD, Nottingham Trent University.
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Abstract
Traditional methods of salmonella isolation and detection rely on the growth of an organism to detectable levels, requiring a minimum of 72h to achieve a presumptive salmonella colony.
An antigen-capture enzyme immunoassay was developed in order to provide a more rapid method of salmonella detection. Specific antisera, immobilised onto magnetic beads (Dynabeads* anti-Salmonella) were used to capture and isolate live salmonella cells from traditional pre-enrichment broths. The captured salmonellas were then post-enriched for 6h in GN broth plus novobiocin (nGN) followed by boiling. The heat treated cells were captured by a second batch of magnetic beads and detected by the Salmonella genus specific monoclonal antibody Ml05 in an immunomagnetic separation enzyme immunosorbent assay (IMS-ELISA). The total assay time was under 27h. The sensitivity limit of the IMS-ELISA in pure culture experiments was 105 cfu/ml. The IMS-ELISA detected the commonly isolated Salmonella serogroups B-E.
A preliminary food trial experiment compared salmonella detection of artificially inoculated raw chicken samples by traditional Rappaport Vassiliadis with selective plating on xylose lysine desoxycholate agar (RV-XLD), conventional IMS selective plating (IMS- XLD) and the IMS-ELISA method. Buffered peptone water (BPW)-raw chicken samples were artificially inoculated with five Salmonella strains (S.typhimurium, S.virchow, S.enteritidis, S.ealing and S.arizonae) at three inoculum levels (100, 10 and 1 cfu/ml). Traditional RV-XLD, IMS-XLD and IMS-ELISA methods detected Salmonella spp. in 14/18, 16/18 and 8/18 samples respectively. Salmonella was detected at all inoculum levels with the exception of S.enteritidis one cell inoculum size. Plate inoculation of nGN broths confirmed that the high number of false negative results for the IMS-ELISA was due to failure of salmonella numbers to reach the detection limit of the IMS-ELISA.
In addition the reactivity of LPS isolated from 42 Salmonella strains and 12 nonsalmonella Enterobacteriaceae with the antibody Ml05 was analysed. Immunoblot assay of SDS-PAGE separated LPS molecules revealed that the Ml05 antibody reacted with (at the very least) the R-type LPS of 40 out of the 42 Salmonella strains tested. The lack of reaction of S.djakarta and a strain of S.arizonae (reference number 1803) implies an absence of the M105 epitope. Hence it is plausible that atypical salmonella core structures exist.
| Item Type: | Thesis | ||||
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| Creators: | Mansfield, L.P. | ||||
| Date: | 1999 | ||||
| ISBN: | 9781369325386 | ||||
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| Divisions: | Schools > School of Science and Technology | ||||
| Record created by: | Linda Sullivan | ||||
| Date Added: | 28 Jun 2021 11:31 | ||||
| Last Modified: | 13 Dec 2023 14:49 | ||||
| URI: | https://irep.ntu.ac.uk/id/eprint/43262 |
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