Research article Isolation of scFv fragments specific to OmpD of Salmonella Typhimurium Torsten Meyer a , Janin Stratmann-Selke b , Jochen Meens c , Thomas Schirrmann a , Gerald F. Gerlach b,c , Ronald Frank d , Stefan Du ¨ bel a , Katrin Strutzberg-Minder b , Michael Hust a, * a Technische Universita ¨t Braunschweig, Institut fu ¨r Biochemie und Biotechnologie, Abteilung Biotechnologie, Spielmannstr. 7, 38106 Braunschweig, Germany b IVD GmbH Heisterbergallee 12, 30453 Hannover, Germany c Stiftung Tiera ¨rztliche Hochschule Hannover, Institut fu ¨r Mikrobiologie, Zentrum fu ¨r Infektionsmedizin, Bischofsholer Damm 15, 30173 Hannover, Germany d Helmholz Zentrum fu ¨r Infektionsforschung, Chemische Biologie, Mascheroder Weg 1, 38124 Braunschweig, Germany 1. Introduction Salmonella spec. is a genus of the Enterobacteriaceae, and today more than 2400 serotypes are known. More than 1400 serotypes belong to the subspecies Salmonella enterica subspecies enterica (Le Minor et al., 1986). The serotype Typhimurium is not host adapted and can infect both animals and humans (Thorns, 2000) and represents the most isolated serotype in industrial countries. In swine, the main isolate is phagetype Salmonella Typhimurium DT104. Human infections with this strain are very critical, because it carries resistances to most of the commonly used anti- biotics. Established methods for Salmonella Typhimurium diagnostic are classically time-intensive, using microbiolo- gical cultures on different liquid and solid media (Farmer, 1999; Gaillot et al., 1999), the use of specific fluorescence labeled DNA probes (Silverman and Kool, 2005), or PCR (Alvarez et al., 2004). ELISA is the method of choice for the diagnostic of Salmonella Typhimurium in high throughput (Nielsen et al., 1995; Steinbach and Staak, 2001). The outer membrane protein D (OmpD) is one of the major proteins of the outer membrane of Salmonella Typhimurium and is described to be immunogenic (Hust et al., 2006). It is a 34 kDa porin and is expressed in addition to OmpF and OmpC (Nikaido and Vaara, 1985; Singh et al., 1992). Salmonella Typhi does not have the OmpD (Santiviago et al., 2001). OmpD is very homologous to NmpC and Lc porin of Escherichia coli K12 (Singh et al., 1992). It is involved in the resistance of non-typhoid Salmonella to methyl-viologen (Santiviago et al., 2002). One group described an OmpD mutant strain showing a decreased virulence (Dorman et al., 1989) while other Veterinary Microbiology 147 (2011) 162–169 ARTICLE INFO Article history: Received 2 February 2010 Received in revised form 26 May 2010 Accepted 23 June 2010 Keywords: Salmonella Typhimurium OmpD Competitive ELISA, scFv Pig Recombinant antibody ABSTRACT Pork meat is one of the major sources for human infections with Salmonella enterica subspecies enterica serovars. Further, zoonoses caused by S. enterica subspecies enterica serovars are responsible for substantial economical losses in industrial countries. Quick and reliable detection of this infection is urgently needed to improve consumer security. Due to its capability to identify infections independent of the species, a competitive ELISA is the preferable method for the detection of anti-Salmonella antibodies in serum. Recombinant antibody fragments (scFvs) were isolated from the naive human antibody gene library HAL7 by phage display. Recombinant produced outer membrane protein D (OmpD) of Salmonella Typhimurium was used as antigen. The characterization of the isolated single chain Fv (scFv) antibodies was done by enzyme-linked immunosorbent assay (ELISA), immunoblot, sequencing, epitope mapping and size exclusion chromatography (SEC). The detection of anti-OmpD IgGs in swine sera by competitive ELISA was shown in a proof of principle concept. Furthermore, the developed competitive ELISA would be compatible to a recently published DIVA vaccine, allow to distinguish between infected and vaccinated pigs. ß 2010 Elsevier B.V. All rights reserved. * Corresponding author. Tel.: +49 531 391 5760; fax: +49 531 391 5763. E-mail address: m.hust@tu-bs.de (M. Hust). Contents lists available at ScienceDirect Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic 0378-1135/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2010.06.023