Antibody Immobilization on Waveguides Using a Flow–Through System Shows Improved Listeria monocytogenes Detection in an Automated Fiber Optic Biosensor: RAPTOR™

Recent outbreaks of food borne illnesses continue to support the need for rapid and sensitive methods for detection of foodborne pathogens. A method for detecting Listeria monocytogenes in food samples was developed using an automated fiber-optic-based immunosensor, RAPTOR™. Detection of L. monocytogenes in phosphate buffered saline (PBS) was performed to evaluate both static and flow through antibody immobilization methods for capture antibodies in a sandwich assay. Subsequent detection in frankfurter samples was conducted using a flow through immobilization system. A two stage blocking using biotinylated bovine serum albumin (b-BSA) and BSA was effectively employed to reduce the non-specific binding. The sandwich assay using static or flow through mode of antibody immobilization could detect 1×10(3) cfu/ml in PBS. However, the effective disassociation constant K(d) and the binding valences for static modes of antibody immobilization in spiked PBS samples was 4×10(5)cfu/ml and 4.9 as compared to 7×10(4) cfu/ml and 3.9 for flow through method of antibody immobilization. Thus the sensitive flow-through immobilization method was used to test food samples, which could detect 5×10(5)cfu/ml of L. monocytogenes in frankfurter sample. The responses at the lowest detectable cell numbers in the frankfurter samples was 92.5 ± 14.6 pA for L. monocytogenes to comparative responses of 27.9 ± 12.2 and 31 ± 14.04 pA obtained from Enterococcus faecalis and Lactobacillus rhamnosus (control species), respectively. The effective K(d) and binding valency from spiked frankfurter samples was 4.8×10(5) cfu/ml and 3.1, thus showing highly sensitive detection can be achieved using the RAPTOR™ biosensor even in the presence of other bacterial species in the matrix.