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United States Department of Agriculture

Agricultural Research Service

Research Project: INTEGRATED BIOSENSOR-BASED PROCESSES FOR MULTIPATHOGENIC ANALYTE DETECTION

Location: Molecular Characterization of Foodborne Pathogens

Title: Spr Biosensor for the Detection of L. Monocytogenes Using Phage Displayed Antibody

Authors
item Nanduri, Viswaprakash - PURDUE UNIVERSITY
item PAOLI, GEORGE
item BREWSTER, JEFFREY
item Bhunia, Arun - PURDUE UNIVERSITY
item TU, SHU-I

Submitted to: Biosensors and Bioelectronics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 5, 2007
Publication Date: N/A

Interpretive Summary: Food production facilities and regulatory inspectors need sensitive and accurate tests for the detection of harmful bacteria (pathogens) in food and on food processing surfaces. Rapid tests are essential to allow detection of contaminated foods before they are distributed to consumers. Tests that can be conducted in the field are especially attractive as they eliminate the delay caused by transporting samples to the laboratory. Rapid tests require antibodies (the same molecules produced in the immune system to clear infectious agents from the body) that bind tightly and specifically to the target pathogen. L. monocytogenes is of particular interest, since it is able to grow at refrigerator temperatures and has a high fatality rate in infected individuals. Tests for L. monocytogenes must be very specific, since other, non-harmful species of Listeria are widely distributed in the environment. In this research we used a new, highly specific antibody to L. monocytogenes developed in our laboratory using phage display technology. This antibody was combined with a compact, inexpensive sensor based on surface-plasmon resonance (SPR) to create a biosensor capable of detecting L. monocytogenes in minutes. A simple adsorption process for fixing the antibody on the sensor surface was used, avoiding costly chemical processes usually used with SPR sensors. This instrument could be configured as a portable, handheld device for use on farms and in processing plants, providing researchers and regulators with an important tool for controlling food-borne disease.

Technical Abstract: 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 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, three-channel SPREETAâ„¢ sensor. Specificity of the sensor was tested using common foodborne 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 5x104 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.

Last Modified: 9/29/2014
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