Background Diarrhea is a prevalent pathological condition frequently associated to the

Background Diarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic (ETEC) strains known to be endemic in developing countries. hybridoma clones. This ongoing work reports their design construction molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence the purified toxins by ELISA and also LT- ST- and LT/ST-producing ETEC strains. Conclusion The developed recombinant scFvs against ST and LT constitute promising starting point for simple and cost-effective ETEC diagnosis. Introduction Up to 5 million cases of diarrhea are reported around the world leading to thousands of deaths per year in children under five years of age [1]. Diarrheagenic (DEC) are the most frequent bacterial etiological agent in particular enterotoxigenic (ETEC) which is endemic in essentially all developing countries. Also approximately 20 to 60% of travelers to developing countries contract diarrheal disorders being ETEC the etiological agent responsible for most of them [2]. ETEC strains produce colonization factors which allow the organisms to readily colonize the small intestine and in this way leading to diarrhea due to the production of heat-labile (LT) and/or heat-stable (ST) enterotoxins [3 4 5 Since ETEC comprise a wide range of O antigenic types diagnosis must depend upon the detection of LT and ST enterotoxins. As revised and well addressed by Qadri and colleagues several immunoserological assays were established for the detection of ST and LT but regrettably in developing countries there are still no simple readily available tools and/or methods that can be used to identify these organisms in minimally equipped laboratories [6]. Usually serotyping-based diagnosis is the only methodology available in limited-resources settings employing either commercial or in house antisera [7]. For that reason many laboratories conducting studies on the etiology of diarrhea in developing countries do not include ETEC in their routine diagnostic and only research or reference laboratories are skilled to identify these bacteria [6 7 Monoclonal antibodies began to be produced has been widely used presenting various advantages such as easy handling fast growth short time for protein expression simple and inexpensive culture media and high performance. Another factor that contributes to their broad range use is the availability PF-00562271 of a large number of vectors and strains which facilitates the gene cloning and the proteins production [15 16 The convenience of genetic engineering has enabled the development of recombinant antibodies in scFv format against PF-00562271 different antigens of DEC pathotypes that can be used as a tool for diagnosis. Considering that the objective of this work consisted in the production and characterization of scFv molecules to detect LT and ST toxins of ETEC. Methods and materials Ethics statement No animal model was employed in the present work. The hybridomas used as template for scFv development were previously obtained [17 18 for LT monoclonal antibody (mAb) and for ST mAb respectively. All experiments were conducted in agreement with the Ethical Principles in Animal Research adopted by the Brazilian College of Animal Experimentation and they were TSPAN32 approved by the Ethical Committee for Animal Research of Butantan Institute (314/06). Y-1 cells from mouse adrenal gland (ATCCCCL79) and Caco-2 from human colorectal adenocarcinoma (ATCCHTB37) were used in LT and ST cell interaction assays respectively. Bacterial strains and plasmids The following K12 strains were used: DH5α (Stratagene USA) BL21 (DE3) (Novagen USA) and C43 (DE3) (Lucigen USA). The plasmid vector pET28a was obtained from Novagen (USA) and the pGEM-T Easy Vector System kit from Promega (USA). Bacterial isolates used in this study consisted of strains previously defined as ETEC by the presence of LT and/or ST encoding-gene as well as the production of the respective toxins [18]. Also ETEC “type”:”entrez-nucleotide” attrs :”text”:”H10407″ term_id :”875229″ term_text :”H10407″H10407 (O78:H11) and 3321–4 (O153:H45) were employed as PF-00562271 ST/LT-producing and ST-producing prototypes respectively [19 20 PCR analyses for toxins types Primer design Alignment of multiple available sequences of PF-00562271 (LTI) from GenBank ({“type”:”entrez-nucleotide” attrs :{“text”:”NC_014232″ term_id.