title: Monoclonal antibodies against all known variants of EspA: development of a simple diagnostic test for enteropathogenic Escherichia coli based on a key virulence factor.
abstract: Enteropathogenic Escherichia coli (EPEC) are a major cause of infant diarrhoea in developing countries and a significant public health issue in industrialized countries. Currently there are no simple tests available for the diagnosis of EPEC. Serology of O-antigens is widely used routinely in many laboratories throughout the world, even though it has been known for many years to be an unreliable indicator of EPEC virulence. We have developed a simple, low-cost immunodiagnostic test based on the EspA filament, an essential virulence factor of EPEC and the related enterohaemorrhagic E. coli (EHEC). Using recombinant proteins of the five major variants of EspA as immunogens, we raised a panel of three monoclonal antibodies in mice that detects all variants of the native target in bacterial cultures. The antibodies proved suitable for application in sandwich-type assays, including ELISA and lateral flow immunoassays (LFI). Prototypes for both assays were specific for EPEC and EHEC strains when tested against a panel of control micro-organisms. We have also developed a simple, affordable culture medium, A/E medium, which optimizes expression of EspA allowing improved sensitivity of detection compared with standard Dulbecco's modified Eagle's medium. Together these reagents form the basis of robust, informative tests for EPEC for use especially in developing countries but also for routine screening in any clinical laboratory.
authors.affiliation: Department of Genetics, University of Leicester, Leicester, UK.
authors.forename: Uta
authors.initials: U
authors.lastname: Praekelt
authors.affiliation: Abteilung für Infektionskrankheiten, Robert Koch Institut, Wernigerode, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Reissbrodt
authors.affiliation: Abteilung für Infektionskrankheiten, Robert Koch Institut, Wernigerode, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kresse
authors.affiliation: Centre for Biotechnology, Anna University, Chennai, India.
authors.forename: Asalapuram
authors.initials: A
authors.lastname: Pavankumar
authors.affiliation: Centre for Biotechnology, Anna University, Chennai, India.
authors.forename: Krishnan
authors.initials: K
authors.lastname: Sankaran
authors.affiliation: Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, UK.
authors.forename: Roger
authors.initials: R
authors.lastname: James
authors.affiliation: Christian Medical College, Vellore, India.
authors.forename: Mary
authors.initials: M
authors.lastname: Jesudason
authors.affiliation: Christian Medical College, Vellore, India.
authors.forename: Shalini
authors.initials: S
authors.lastname: Anandan
authors.affiliation: Christian Medical College, Vellore, India.
authors.forename: Agila
authors.initials: A
authors.lastname: Prakasam
authors.affiliation: Christian Medical College, Vellore, India.
authors.forename: Veeraraghavan
authors.initials: V
authors.lastname: Balaji
authors.affiliation: National Institute of Cholera and Enteric Diseases, Kolkata, India.
authors.forename: Shanta
authors.initials: S
authors.lastname: Dutta
authors.affiliation: National Institute of Cholera and Enteric Diseases, Kolkata, India.
authors.forename: Sanjucta
authors.initials: S
authors.lastname: Dutta
authors.affiliation: National Institute of Cholera and Enteric Diseases, Kolkata, India.
authors.forename: Thandavarayan
authors.initials: T
authors.lastname: Ramamurthy
authors.affiliation: R-Biopharm AG, Darmstadt, Germany.
authors.forename: Renate
authors.initials: R
authors.lastname: Fischer
authors.affiliation: R-Biopharm AG, Darmstadt, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Sander
authors.affiliation: Institut für Medizinische Mikrobiologie, Universität Leipzig, Leipzig, Germany.
authors.forename: Reiner
authors.initials: R
authors.lastname: Schaumann
authors.affiliation: Department of Public Health, Faculty of Medicine, National Autonomous University of Mexico, Mexico City, Mexico.
authors.forename: Armando
authors.initials: A
authors.lastname: Navarro
authors.affiliation: Department of Genetics, University of Leicester, Leicester, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Williams
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D003955:Diagnostic Tests, Routine / Q000379:methods*; D054324:Enterohemorrhagic Escherichia coli / Q000276:immunology / Q000302:isolation & purification*; D054308:Enteropathogenic Escherichia coli / Q000276:immunology / Q000302:isolation & purification*; D004927:Escherichia coli Infections / Q000175:diagnosis* / Q000382:microbiology; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000276:immunology; D005759:Gastroenteritis / Q000175:diagnosis* / Q000382:microbiology; D006801:Humans; D007159:Immunologic Tests / Q000379:methods; D008807:Mice, Inbred BALB C; D037521:Virulence Factors / Q000032:analysis / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D029968:Escherichia coli Proteins; C102051:EspA protein, E coli; D037521:Virulence Factors