B11003: Diagnostic application of monoclonal antibody-based sandwich ELISAs for the rapid detection of verocytotoxin-producing E. coli

Study Duration: August 2000 to November 2003

Contractor: The Queen's University of Belfast

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. 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. 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. 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. 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. Objectives of the project include:

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.

2. Determination of the optimum application of the MAbs by investigation of various ELISA methods and by combining ELISA stages with enrichment.

3. Using selected assays to survey animal and human clinical isolates, potential food sources for human infection and comparison with existing approved assays.

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.

1. Completing the development of a specific E. coli O157 assay:

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.

2. Optimal application of the sandwich ELISA, using O157 broth cultures and spiked faecal samples:

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.

3. Application of selected assay(s) in surveys:

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.

None of the new monoclonal antibodies (MAbs) raised provided suitable reagents for the provision of an E. coli O157 specific sELISA. Most of them were produced and purified in prospectively suitable concentrations, but the major problem appeared to be the lack of a consistent expression of the target antigen. Positive sandwich enzyme linked immunosorbent assay (sELISA) results were obtained with a combination of MAb 7A6 with some of the intimin MAbs, but these results showed great variations on repeat, with the same O157 strain and between strains. Attempts were made to improve expression by adding to the medium ingredients that have been shown to increase the intimin level, but no assay improvement was obtained.

With the failure of these MAbs to provide suitable Enterohemorrhagic E. coli (EHEC) specific sELISA reagents, other attempts were made to obtain more suitable MAbs. Some success was obtained with sELISA reagents prepared from a polyclonal antibody (PAb) prepared from an EHEC O157 strain and absorbed with a non-EHEC O157 strain. The assay with the absorbed serum showed EHEC O157 specificity when tested against a number of O157 and non-O157 strains. However, the reagents lost their activity following storage at -20°C, and attempts to repeat the absorption with the same PAb and with others was not successful. On the basis that this result indicated the existence of suitable surface antigen to target for EHEC O157 specificity, more hybridoma fusions were carried out with mice immunised with antigens prepared from EHEC O157, including the lipopolysaccharide and the outer membrane proteins. None of these produced suitable MAbs.

With the unavailability of appropriate EHEC O157 specific sELISA reagents, work for the development of a capture/enrichment sELISA protocol was undertaken with MAbs 7A6 and 2F3. The procedure adopted was to incubate a 1 in 10 and a 1 in 100 dilution of the sample in phosphate buffered saline (PBS) in the capture stage for 2 hours at 37°C, wash off the unbound material and add tryptone soya broth for further incubation overnight. The well contents after enrichment were removed for storage before completion of the sELISA. The contents of ELISA positive wells were streaked onto blood agar, cultured and single colonies were further tested by sELISA without enrichment to identify those for isolation of pure cultures.

This procedure was demonstrated to have, at its best, a sensitivity of a <10 colony forming units (cfu) per ml for MAb 7A6 and 10² cfu per ml for MAb 2F3. In a survey carried out with the MAb 7A6 sELISA, 20 sELISA positive reactions were detected out of 400 bovine faeces samples from enteritis cases, submitted to the Institute for diagnostic investigation. ELISA positive samples were cultured on sorbital McConkey agar, but no EHEC O157 was detected. Immunomagnetic bead separation (IMS) was also carried out on the same samples and no EHEC O157 was detected by this procedure either. This result illustrates the problems afforded by the comparatively higher incidence of non-EHEC to EHEC O157 strains in bovine faeces, and the unavailability of EHEC O157 specific MAbs.

Similar surveys were carried out with the MAb 2F3 sELISA. In one, 21 ELISA positive reactions were detected out of 422 bovine faeces samples from enteritis cases, and pure O26 culture isolates were obtained from 12 of the ELISA positive samples. This number of strains isolated in pure culture from sELISA positive samples by this procedure is more than has been obtained in previous non-enrichment sELISA work with this MAb, from re-culture of sELISA positive samples from the original faeces sample.

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).

Contact: For any enquiries concerning this research project, please contact the relevant programme contact or email science@foodstandards.gsi.gov.uk