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AEEC INFECTIONS Résumé de rapport

Project ID: QLK2-CT-2000-00600
Financé au titre de: FP5-LIFE QUALITY
Pays: United Kingdom

Development of immuno-capture and ELISA diagnostic tests

The main serotype implicated in human disease caused by EHEC strains has been O157 but other serotypes have also been implicated in causing human and/or animal diseases. The main objective of this WP is to develop diagnostic tests for the detection of non-O157 EHEC in particular and AEEC in general.

We will develop two different types of test:
- The first type of diagnostic test will be based on immuno-magnetic separation (IMS) for selective enrichment of AEEC strains. Theses IMS tests will use polyclonal (already developed by partner CR2) and monoclonal (to be developed by partner CR10) antibodies directed against intimin;

- The second type of test will be sandwich ELISA tests for characterisation of AEEC strains. The ELISA will be based on intimin Mabs produced against the different intimin types as well as Mabs directed toward surface adhesin (see WP16).

Major finding during the project
The primary aim of WP21 was to develop new reagents for isolation and detection of VTEC. Intimin and LPS are well-characterised surface VTEC antigens. Accordingly, mono-specific antibodies against these major antigens were generated and evaluated in this project.

Intimin - several attempts were made to generate monoclonal antibodies using different intimin domains as an antigen. Intimin shows antigenic polymorphism. The carboxy terminus domain, comprising the receptor-binding activity, is variable, yet expose on the bacterial cell surface. The amino terminus is conserved among the different VTEC strains, yet masked by the LPS. Monoclonal antiserum made using the carboxy terminal 280 amino acids of intimin a and intimin b, although reacted with the antigen did not cross-react with the native intimin on the bacterial cell surface. This rendered the antibodies unsuitable for immuno magnetic separation (IMS). Polyclonal antibodies made against the conserved region of intimin bound recognised native intimin, but the binding avidity was too low for IMS. The conclusion from this investigation was that intimin is unlikely to provide a good target for capturing VTEC from biological material and foodstuff or for ELISA.

LPS- is a strong antigen that is constitutively expressed by VTEC. However, VTEC bacteria can expressed one of a number of distinct LPS antigens; accordingly, a number of serogroup-specific antibodies are needed to be used in parallel IMS for each sample. Nevertheless, as the conserved intimin domain was proven unsuitable for a pan VTEC detection/isolation system, we evaluated the use of LPS-specific antisera. Antibodies directed against O26, O103 and O111 and O157 were evaluated. Using the antisera in reconstituted biological material and in field studies, LPS antibodies were proven to be highly specific and sensitive in IMS.

Conclusion - at present, a bank of serogroup-specific LPS antibodies are the recommended method for IMS of VTEC.

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