SPR biosensor for the detection of L. monocytogenes using phag4-displyed antibody

Center For Food Safety and Engineering, Department of Food Science, Purdue University, 745 Agriculture Mall Dr., West Lafayette, IN 47907, USA.
Biosensors & Bioelectronics (Impact Factor: 6.41). 10/2007; 23(2):248-52. DOI: 10.1016/j.bios.2007.04.007
Source: PubMed


Whole cells of Listeria monocytogenes were detected with a compact, surface plasmon resonance (SPR) sensor using a phage-displayed scFv antibody to the virulence factor actin polymerization protein (ActA) for biorecognition. Phage Lm P4:A8, expressing the scFv antibody fused to the pIII surface protein was immobilized to the sensor surface through physical adsorption. A locally constructed fluidics system was used to deliver solutions to the compact, two-channel SPREETA sensor. Specificity of the sensor was tested using common food-borne bacteria and a control phage, M13K07 lacking the scFv fusion on its coat protein. The detection limit for L. monocytogenes whole cells was estimated to be 2 x 10(6)cfu/ml. The sensor was also used to determine the dissociation constant (Kd) for the interaction of phage-displayed scFv and soluble ActA in solution as 4.5 nM.

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Available from: Viswaprakash Nanduri
    • "There are several types of bacteriophage, such as T4, Lambda and lytic phage; however, a filamentous phage called M13 has been extensively used for phage display technology, since it was discovered in 1985 that foreign DNA fragments could be inserted to create a fusion protein [15]. Later, phage-displayed antibody libraries were constructed by cloning antibody-binding fragments into a phage gene and these have been employed in various applications [16] [17] [18] [19] [20]. In addition to phage-displayed antibodies, the filamentous bacteriophage libraries displaying short peptides or proteins have been used for different purposes such as discovery of protein targets [21] [22], organic compound target [23], and epitope identification for vaccine development [24]. "
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    ABSTRACT: Surface plasmon resonance (SPR)-based biosensor is a popular platform for real-time monitoring and sensitive detection for a myriad of targets. However, only a few studies have reported the use of bacteriophages as specific binders for SPR-based detection. This study aimed to demonstrate how filamentous M13 bacteriophages expressing 12-mer peptides can be employed in an SPR-based assay, using a Salmonella-specific bacteriophage as a model binder to detect the foodborne bacterium Salmonella. Several important factors (immobilization buffers and methods, and interaction buffers) for a successful bacteriophage-based SPR assay were optimized. As a result, a Salmonella-specific bacteriophage-based SPR assay was achieved, with very low cross reactivity with other non-target foodborne pathogens and detection limits of 8.0 x 10(7) and 1.3 x 10(7) CFU/mL for one-time and five-time immobilized sensors, respectively. This proof-of-concept study demonstrates the feasibility of using M13 bacteriophages expressing target-specific peptides as a binder in a rapid and label-free SPR assay for pathogen detection.
    No preview · Article · Jan 2014 · Sensors and Actuators B Chemical
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    • "Phage display biopanning is a technique extending from the invention of phage display technology in 1985 [22] and offers an alternative means of generating specific affinity ligands. This technique has been used previously in an attempt to generate alternative binders to L. monocytogenes; Paoli et al. [5,23] and Nanduri et al. [24] produced phage display-derived antibody fragments to L. monocytogenes, while Carnazza et al. [25] described the production of phage display-derived peptide binders to L. monocytogenes. However, Carnazza et al. presented no data on cross-reactivity of their binders with other Listeria spp. or relevant foodborne bacteria to support the specificity claim. "
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    ABSTRACT: The objective of this study was to produce phage display-derived binders with the ability to distinguish Listeria monocytogenes from other Listeria spp., which may have potential utility to enhance detection of Listeria monocytogenes. To obtain binders with the desired binding specificity a series of surface and solution phage-display biopannings were performed. Initially, three rounds of surface biopanning against gamma-irradiated L. monocytogenes serovar 4b cells were performed followed by an additional surface biopanning round against L. monocytogenes 4b which included prior subtraction biopanning against gamma-irradiated L. innocua cells. In an attempt to further enhance binder specificity for L. monocytogenes 4b two rounds of solution biopanning were performed, both rounds included initial subtraction solution biopanning against L. innocua. Subsequent evaluations were performed on the phage clones by phage binding ELISA. All phage clones tested from the second round of solution biopanning had higher specificity for L. monocytogenes 4b than for L. innocua and three other foodborne pathogens (Salmonella spp., Escherichia coli and Campylobacter jejuni). Further evaluation with five other Listeria spp. revealed that one phage clone in particular, expressing peptide GRIADLPPLKPN, was highly specific for L. monocytogenes with at least 43-fold more binding capability to L. monocytogenes 4b than to any other Listeria sp. This proof-of-principle study demonstrates how a combination of surface, solution and subtractive biopanning was used to maximise binder specificity. L. monocytogenes-specific binders were obtained which could have potential application in novel detection tests for L. monocytogenes, benefiting both the food and medical industries.
    Full-text · Article · Sep 2013 · PLoS ONE
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    • "Rapid and reliable detection methods of this pathogenic, toxin-producing bacterium are required since it is able to survive and grow at low temperatures [22] and because the mortality rate for infected individuals is much higher than for other common foodborne pathogens [23] [24] [25] [26] [27]. "
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    ABSTRACT: Internalin A (InlA), a protein required for Listeria monocytogenes virulence, is encoded by the inlA gene, which is only found in pathogenic strains of this genus. One of the best ways to detect and confirm the pathogenicity of the strain is the detection of one of the virulence factors produced by the microorganism. This paper focuses on the design of an electrochemical genosensor used to detect the inlA gene in Listeria strains without labelling the target DNA. The electrochemical sensor was obtained by immobilising an inlA gene probe (single-stranded oligonucleotide) on the surfaces of screen-printed gold electrodes (Au-SPEs) by means of a mercaptan-activated self-assembled monolayer (SAM). The hybridisation reaction occurring on the electrode surface was electrochemically transduced by differential pulse voltammetry (DPV) using methylene blue (MB) as an indicator. The covalently immobilised single-stranded DNA was able to selectively hybridise to its complementary DNA sequences in solution to form double-stranded DNA on the gold surface. A significant decrease of the peak current of the voltammogram (DPV) upon hybridisation of immobilised ssDNA was recorded. Whole DNA samples of L. monocytogenes strains could be discriminated from other nonpathogenic Listeria species DNA with the inlA gene DNA probe genosensor.
    Full-text · Article · Mar 2013
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