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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Poultry Microbiological Safety and Processing Research Unit » Research » Publications at this Location » Publication #327421

Title: Use of recombinant salmonella flagellar hook protein (flgk) for detection of anti-salmonella antibodies in chickens by automated capillary immunoassay

item Yeh, Hung-Yueh
item ACOSTA, AIMEE - University Of Puerto Rico
item SERRANO, KATHERINE - University Of Puerto Rico
item Buhr, Richard - Jeff

Submitted to: American Society for Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 2/29/2016
Publication Date: N/A
Citation: N/A

Interpretive Summary: none

Technical Abstract: Background: Conventional immunoblot assays are a very useful tool for specific protein identification, but are tedious, labor-intensive and time-consuming. An automated capillary electrophoresis-based immunoblot assay called "Simple Western" has recently been developed that enables the protein separation, blotting and detection in an automatic manner. This technology has recently been used by pharmaceutical industries to identify, evaluate and characterize biophamarceutical products. However, this technology has not been used in clinical diagnosis. The flagellar hook protein (FlgK) is required for flagellar filament formation. Further, FlgK is an important virulence factor that plays a role in intestinal inflammation and is the most immune-reactive flagellar protein. Therefore, it is a potential target for host immune response. In this communication, we evaluated whether the Simple Western system could be used to detect Salmonella FlgK antibodies from chicken sera. Methods: The recombinant Salmonella FlgK (rFlgK) protein was cloned, expressed and purified using commercial kits. The purity of the protein were determined by SDS-PAGE. Chickens were immunized with rFlgK emulsified in Freund's incomplete adjuvant at the ages of one and three weeks. Appropriate controls were included. Immunoblot analyses were performed in two ways: (1) conventional Western blot analysis as described previously, and (2) Simple Western immunoblot analysis according to the manufacturer's instructions. Chicken sera were also collected from field. Results: The rFlgK protein reacted strongly to sera from the immunized chickens by both conventional Western blot analysis and Simple Western analysis. Sera from un-immunized chickens and commercial specific pathogen free chickens did not react rFlgK. Further, we tested 66 individual chicken sera collected from a single flock raised similar to commercial conditions, and observed 63 out of 66 sera reacted at various degrees to the rFlgK protein by both methods. Conclusion: The Salmonella FlgK protein was successfully expressed in and purified from the E. coli expression system. This protein induced strong immune response in chickens. The Simple Western immunoassay was successfully used for detection of antibodies against the Salmonella rFlgK protein from chicken sera. These results provide a rational for further evaluation of the Simple Western immunoassay as a tool using Salmonella rFlgK in Salmonella outbreak investigation.