Increasing the sensitivity of Listeria monocytogenes assays: evaluation using ELISA and amperometric detection.
An immunosensor for the detection of Listeria monocytogenes was developed. ELISA and amperometric studies were run in parallel to develop a more sensitive and rapid assay for the bacterium. Conditions for the immunosensor were primarily characterised using ELISA. A direct sandwich assay was employed and the affinities of two polyclonal (goat and rabbit) and one monoclonal (mouse) anti-L. monocytogenes antibodies were compared using this format. Owing to low sensitivity being obtained with all antibodies, biotin-avidin amplification and an indirect sandwich assay were employed. The system was then transferred to screen-printed electrodes (SPEs), the primary antibody being immobilised by cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and the mode of detection being amperometric. Various parameters (limit of detection, working range, incubation time, cross-reactivity) of the systems were characterised. The effect of direct incubation in milk is also discussed. The final immunosensor had a working range of 1 x 10(6)-1 x 10(3) cells ml-1 and a detection limit of 9 x 10(2) cells ml-1. The assay took about 3.5 h to complete.
Crowley
E L
EL
Laboratory of Sensor Development, National University of Ireland Cork, Ireland.
O'Sullivan
C K
CK
Guilbault
G G
GG
D015374:Biosensing Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008089:Listeria monocytogenes / Q000302:isolation & purification*