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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 #327122

Title: Detection of anti-salmonella flgk 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: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/10/2016
Publication Date: N/A
Citation: N/A

Interpretive Summary: none

Technical Abstract: Western blot is a very useful tool to identify specific protein, but is tedious, labor-intensive and time-consuming. An automated "Simple Western" assay has recently been developed that enables the protein separation, blotting and detection in an automatic manner. 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. Salmonella FlgK (rFlgK) was expressed and purified using commercial kits. The purity of rFlgK was 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) traditional Western blot analysis as described previously, and (2) Simple Western immunoblot analysis according to the manufacturer's instructions. Chicken sera were also collected from a single flock raised similar to commercial conditions. 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 similar commercial settings, and observed 63 out of 66 sera reacted at various degrees to the rFlgK protein by both methods. Salmonella FlgK was successfully expressed in and purified from the E. coli expression system. This rFlgK protein induced strong immune response in chickens. This automated immunoassay was successfully used for detection of antibodies in chicken sera against Salmonella rFlgK. These results provide a rational for further evaluation of this automated immunoassay as a tool using Salmonella rFlgK in Salmonella epidemiological investigation.