Analytica Chimica Acta 435 (2001) 255–263 Genetically fused single-chain anti-Salmonella antibody with aequorin: a bioluminescence immunoassay for a Salmonella antigen Jianquan Wang a , C. Mark Ensor a , Ginette J. Dubuc b , Saran A. Narang b , Sylvia Daunert a,∗ a Departments of Chemistry and Pharmaceutical Sciences, University of Kentucky, Lexington, KY 40506-0055, USA b Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ont., Canada K1A0R6 Received 28 August 2000; received in revised form 11 January 2001; accepted 25 January 2001 Abstract We report the development of a competitive heterogeneous immunoassay for BSA-conjugated Salmonella antigen using a fusion protein between an anti-Salmonella single-chain antibody (scFv) and the photo-protein aequorin (AEQ). It has been demonstrated that the fusion protein (scFvLAEQ) maintains both the antibody binding affinity toward the Salmonella antigen as well as the luminescence properties of the photoprotein. Using scFvLAEQ, the competitive immunoassay gave a detection limit of 10 g ml -1 of Salmonella antigen. The assay eliminates the need for a labeled secondary antibody usually required in enzyme-linked immuosorbent assays (ELISAs), and therefore, it may find potential applications in detecting Salmonella contamination. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Immunoassay; Single-chain antibody; Salmonella detection; Bioluminescence; Aequorin 1. Introduction Salmonella contamination in food accounts for almost half of the gastrointestinal infections in the United States alone each year. Conventional methods for the detection of Salmonella typically involve sev- eral steps. First, the sample is grown for selection of Salmonella in the culture media. Then, Salmonella needs to be isolated to eliminate potential interfering species. Finally, a variety of serological and biochem- ical tests, such as enzyme immunoassays (EIAs) and enzyme-linked immunosorbent assays (ELISAs) are ∗ Corresponding author. Tel.: +1-859-257-7060; fax: +1-859-323-1069. E-mail address: daunert@pop.uky.edu (S. Daunert). performed to verify the presence of Salmonella [1,2]. Many studies have been done to improve each of these steps with the ultimate goal being the development of a fast and sensitive method for the Salmonella antigen [3,4]. Until recently, radioimmunoassays have been the technique of choice when maximum sensitivity in a bioanalytical assay is required, but public safety concern and regulatory restrictions have stimulated researchers to explore new alternative methods that eliminate the use of hazardous radioisotopes while enabling sufficient sensitivity for detection. One such alternative is ELISA. Different types of ELISA exist depending on the target analyte [5]. For example, in a typical sandwich ELISA for an antigen, a primary antibody is first immobilized onto a solid surface; 0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII:S0003-2670(01)00852-2