Abstract
BackgroundImmunomagnetic separation (IMS) and immunoassays are widely used for pathogen detection. However, novel technology platforms with highly selective antibodies are essential to improve detection sensitivity, specificity and performance. In this study, monoclonal antibodies (MAbs) against Internalin A (InlA) and p30 were generated and used on paramagnetic beads of varying diameters for concentration, as well as on fiber-optic sensor for detection.ResultsAnti-InlA MAb-2D12 (IgG2a subclass) was specific for Listeria monocytogenes and L. ivanovii, and p30-specific MAb-3F8 (IgM) was specific for the genus Listeria. At all bacterial concentrations (103–108 CFU/mL) tested in the IMS assay; the 1-μm diameter MyOne beads had significantly higher capture efficiency (P < 0.05) than the 2.8-μm diameter M-280 beads with both antibodies. The highest capture efficiency for MyOne-2D12 (49.2% for 105 CFU/mL) was significantly higher (P < 0.05) than that of MyOne-3F8 (16.6 %) and Dynabeads anti-Listeria antibody (9 %). Furthermore, capture efficiency for MyOne-2D12 was highly specific for L. monocytogenes and L. ivanovii. Subsequently, we captured L. monocytogenes by MyOne-2D12 and MyOne-3F8 from hotdogs inoculated with mono- or co-cultures of L. monocytogenes and L. innocua (10–40 CFU/g), enriched for 18 h and detected by fiber-optic sensor and confirmed by plating, light-scattering, and qPCR assays. The detection limit for L. monocytogenes and L. ivanovii by the fiber-optic immunosensor was 3 × 102 CFU/mL using MAb-2D12 as capture and reporter antibody. Selective media plating, light-scattering, and qPCR assays confirmed the IMS and fiber-optic results.ConclusionsIMS coupled with a fiber-optic sensor using anti-InlA MAb is highly specific for L. monocytogenes and L. ivanovii and enabled detection of these pathogens at low levels from buffer or food.
Highlights
Immunomagnetic separation (IMS) and immunoassays are widely used for pathogen detection
In the Western blot, monoclonal antibodies (MAbs)-2D12 reacted with an 80-kDa protein band (InlA) from L. monocytogenes and L. ivanovii, but it did not react with other Listeria spp., including L. marthii or L. rocourtiae (Figure 2a)
The resulting MAbs were employed in IMS to capture and concentrate bacteria from food followed by fiber-optic sensor-based detection
Summary
Immunomagnetic separation (IMS) and immunoassays are widely used for pathogen detection. The foodborne pathogen Listeria monocytogenes causes listeriosis—a severe illness that ranges from mild gastroenteritis to invasive infection in immunocompromised people, neonates, and the elderly [1]. In pregnant women, it causes premature births, miscarriages, and neonatal sepsis or fetal deaths. L. monocytogenes is ubiquitous and found in food-processing environments [2,3] and food products, including ethnic soft cheese [4,5], sliced lunch meats [6] and frankfurters, and seafood [7] It has been implicated in numerous food outbreaks and recalls, including a large outbreak involving cantaloupe in the US, which caused 29 deaths and 1 miscarriage [8]. Rapid and accurate detection methods may alleviate some of these problems
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