An official website of the United States government.
The main objective is to develop and optimise a rapid immunologically-based method for the detection and partial characterisation of all VTECs, which are of importance in human illness.
<p>The method is based on dipstick technology whereby the dipstick will be coated with several antibodies able to detect the presence of Verocytotoxin VT1, VT2, somatic antigen of serogroup O157 and the attaching and effacing factor.
<p>The presence of these factors will not only permit the screening of foodstuffs for the presence of all VTECs but will also permit partial characterisation of strains, which will aid the epidemiology of VTEC.
<p>The method will be semi-rapid although it is anticipated that some culture stage will be required. Importantly, it will be suitable for use by surveillance laboratories.
<p>The project aims to fulfil the recommendations of the Advisory Committee on the Microbiological Safety of Food (ACMSF) who, in their 1995 report on VTEC, recommended that the Government fund research into the development of a rapid method for the detection of VTEC in foods and clinical samples.
<p>Objectives of the project include:<BR>
<p>1. Development of an O157 intimin monoclonal antibody (MAb) to be used in a sandwich Enzyme-Linked Immunosorbent Assay (ELISA) in combination with the O157 lipopolysaccharide to produce a VTEC O157 specific assay.
<p>2. Determination of the optimum application of the MAbs by investigation of various ELISA methods and by combining ELISA stages with enrichment.
<p>3. Using selected assays to survey animal and human clinical isolates, potential food sources for human infection and comparison with existing approved assays.
<p>4. Using the information obtained from such assays to contribute to a better understanding of the zoonotic significance of animal O157 carriage for human infection.
<p>1. Completing the development of a specific E. coli O157 assay: <BR>
<p>The development of an O157 intimin MAb will be completed. Uncloned hybridomas are available from a previous hybridoma fusion to a mouse immunised with recombinant beta2-intimin from an O111 VTEC strain. These may produce a MAb that reacts with O157 intimin, but which is unlikely to be specific for O157 strains. In addition, a mouse has been immunised with an O157 recombinant intimin antigen for the eventual development of a specific beta1-intimin MAb. The application of an O157 specific intimin MAb, either at the capture or the development stage of a sandwich ELISA, would contribute to provision of a definitive diagnostic assay for VTEC O157 strains.
<p>2. Optimal application of the sandwich ELISA, using O157 broth cultures and spiked faecal samples:<BR>
<p>Also available for examination at this stage of the project are a polyclonal rabbit serum raised against a peptide sequence that is common to all the currently recognised intimin sub-divisions, recombinant O157 tir antigen, and an O157 lipopolysaccharide MAb that was produced in the previous Department of Health (DH) project. The use of all these reagents that are already available and those that will be produced in the first stage of the project will be examined in various combinations at various ELISA stages. Sandwich ELISA formats will be examined for the development of a protocol of optimum sensitivity. A variety of methods will be examined, including an investigation of combining enrichment with a stage of the ELISA. The sensitivity of each assay will be tested using spiked specimens, and will be compared with the immunomagnetic bead separation method which is generally regarded as the most sensitive assay that is currently in widespread use. The O157 specificity of the assay(s) will also be monitored by using a variety of characterised strains of Enteropathogenic E. coli (EPEC) / VTEC and other E. coli pathogroup origin.
<p>3. Application of selected assay(s) in surveys:<BR>
<P>The assay(s) will be applied to animal and human clinical isolates, abattoir carcass samples and raw meat products. An estimate of the sensitivity and specificity of the selected assay(s) for the examination of field samples will initially be established by comparing the results with those that are obtained from the immunomagnetic bead separation assay. The ELISA assay(s) should allow for the ready testing of large numbers of field samples. Attempts will be made to isolate ELISA reactive strains from ELISA positive field samples by the subsequent testing of strains purified from these samples and by direct culture or by immunomagnetic bead separation assay. In addition, the assays developed will be compared with commercially available ELISA methods.
<p>Find more about this project and other FSA food safety-related projects at the <a href="http://www.food.gov.uk/science/research/" target="_blank">Food Standards Agency Research webpage</a>.
<p>The final report is available from the FSA Library and Information centre. To obtain a copy, please contact the Enquiry Desk, Dr Elsie Widdowson Library and Information Services, Food Standards Agency (tel: 020 7276 8181/8182 or email: library&info@foodstandards.gsi.gov.uk).