Sensitive and specific detection of enteropathogenic and enterohemorrhagic Escherichia coli using recombinant anti-intimin antibody by immunofluorescence assay Andressa Caravelli a , Daniela E. Luz a , Fernanda B. Andrade a , Claudia T.P. Moraes a , Andrea Q. Maranhão b , Roxane M.F. Piazza a, ⁎ a Laboratório de Bacteriologia, Instituto Butantan, São Paulo, SP, Brazil b Laboratório de Imunologia, Universidade de Brasília, DF, Brazil abstract article info Article history: Received 26 April 2013 Received in revised form 20 August 2013 Accepted 23 August 2013 Available online 2 October 2013 Keywords: EPEC EHEC Intimin Recombinant antibody Detection The main and common virulence factor expressed by enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) is intimin, a 94-kDa outer membrane protein, which is a product of the eae gene, and, thus, an excellent target for the detection of these pathogens. Among the methods for detection of virulence factor expression, immunoassays can be considered the first alternative to either animal use or in vitro culture cells assays, for which polyclonal and/or monoclonal antibodies are raised. In the present work, we evaluated the sensitivity and specificity of an intimin recombinant antibody (scFv-intimin) using immunofluorescence assay. The scFv-intimin detected typical EPEC, atypical EPEC, and EHEC isolates (100% sensitivity) with no detection of eae- isolates (100% specificity). Thus, immunofluorescence is an effective and rapid method, and scFv-intimin, an excellent tool for the diagnosis of diarrhea caused by EPEC and EHEC and also can be employed in case–control epidemiological surveys. © 2013 Elsevier Inc. All rights reserved. Diarrhea is the second leading cause of death in children, accounting for ca. 800 thousand deaths per year worldwide, particularly among infants younger than 5 years (WHO, 2012). This disease is prominent in the developing world with low income, poor hygiene, and inadequate sanitation (WHO, 2012). Diarrhea- genic Escherichia coli (DEC) can be considered one of the most important groups of pathogens involved in diarrheal cases, either acute or persistent (Ochoa et al., 2008). Among them, enteropatho- genic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) are responsible for this situation (Trabulsi et al., 2002). Besides diarrhea, EHEC also causes hemorrhagic colitis and produces severe/fatal renal and neurologic complications as a result of the translocation of Shiga toxins (Stx1 and Stx2) across the intestinal wall (O’Brien et al., 1984). The main and common virulence factor expressed by both pathogens and involved in the attaching/effacing (A/E) lesion is intimin, a 94-kDa outer membrane protein, which is a product of the eae gene (Jerse et al., 1990), and thus, it is an excellent target for the detection of these pathogens. Despite the current efforts in the development of reliable and low-cost diagnostic assays, DEC detection assays are currently unavailable in most developing countries, except for Shiga toxin- producing E. coli (STEC) in certain countries where the other categories are frequently disregarded as important causes of either infantile diarrhea or diarrheal disease in all age groups (Piazza et al., 2010). Thus, the identification of the characteristic virulence genes is an obvious choice for detection by PCR to determine one or multiple virulence genes. Nevertheless, gene detection does not assure expression of the corresponding virulence factor, and besides, the great genetic diversity of intimin, demonstrated by the description of at least 27 subtypes, makes its molecular detection difficult, which depends on the primer sequences used (Blanco et al., 2006; Zhang et al., 2002). Moreover, a negative PCR result may not indicate that intimin is absent. Considering virulence factors expression, one can use either animals or in vitro culture cells, both methods present more disadvantages than advantages, since the use of animals and in vitro culture cells for routine diagnosis are time consuming, laborious, and cumbersome; thus, immunoassays can be considered the first alternative for virulence factors expression, for which polyclonal and/or monoclonal antibodies are raised (Batchelor et al., 1999; Koga et al., 2003; Menezes et al., 2010). Considering EPEC and EHEC diagnosis, the conserved region of this protein (Int 388-667 ) was used as antigen for the development of a potential universal anti-intimin antibody able to detect the current known intimin subtypes and further uncharacterized ones (Batchelor et al., 1999; Koga et al., 2003; Menezes et al., 2010). Also, a monoclonal anti-intimin IgG2b antibody Diagnostic Microbiology and Infectious Disease 77 (2013) 301–303 ⁎ Corresponding author. Tel.: +55-11-26279724. E-mail address: roxane@butantan.gov.br (R.M.F. Piazza). 0732-8893/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2013.08.021 Contents lists available at ScienceDirect Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio