A new technique for amplified immunoassay will be developed for sensitive measurement of bacteria under conditions which do not destroy them, coupled with the isolation of these bacteria from the immunoassay matrices. The isolated bacteria could be characterized in order to assess the epidemiology of infection by the organism, or be tested for their antibiotic resistance. The organisms to be tested in this Phase I effort are E. coli O157/H7, and several Salmonella isolates. The method will include measurement of bacteria via surface LPS antigens by a modified ELISA using fluoresceinated and Russell s viper venom factor X activator-conjugates (RVV-XA-Ab) and capture on an anti- fluorescein matrix, and detected using a coagulation-based assay amplification system known as Enzyme-linked coagulation assay, or ELCA. After measurement, these bound bacteria will be eluted using fluoresceyl gelatin or fluoresceyl casein, counted by quantitative PCR and tested for their viability. Quantitative PCR for Salmonella will use detection of an invE-invA gene and/or and rfbS gene coding for tyvelose synthetase; detec2tion of E. coli O157 will use genes for SLT-I, SLT-II and rfbEO0157:H7. Development of this approach will include the use of microtiter plates and a filter device used to capture bacteria on immunosorbent filters with a pore size of 5 mum which will allow elution of bacteria. The system will be developed to optimize detection/isolation of these bacteria in Phase I, and will be extended to a number of other bacterial species in Phase II. This technique will have considerable value as a more rapid and less expensive approach for isolation of pathogenic bacteria than classical microbiological methods.
The simplified, rapid and sensitive detection of bacteria coupled with immunoisolation will simplify epidemiological studies which depend on isolated bacteria, and will make it possible to more rapidly assess the antibiotic sensitivity of organisms found to be infectious. These features of the approach will be of considerable practical value and will lead to commercially viable assay kits.
