Available online at www.sciencedirect.com Journal of Virological Methods 147 (2008) 235–243 Selection of phage-displayed human antibody fragments on Dengue virus particles captured by a monoclonal antibody: Application to the four serotypes Sheila Cabezas a,b,∗ , Gertrudis Rojas b , Alequis Pavon a , Mayling Alvarez a , Maritza Pupo a , Gerardo Guillen b , Maria G. Guzman a a Department of Virology, PAHO/WHO Collaborating Center for the Study of Dengue and its Vector, “Pedro Kour´ ı” Tropical Medicine Institute, Autopista Novia del Mediod´ ıa, km 6 5, P.O. Box 601 Marianao 13, Habana, Cuba b Pharmaceuticals Division, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Ave 31 e/158 y 190, Playa, La Habana 10600, Cuba Received 30 June 2007; received in revised form 1 September 2007; accepted 5 September 2007 Available online 22 October 2007 Abstract Antibody fragments to the four Dengue virus serotypes were isolated from a human universal na¨ ıve library using phage display technology. Phage-displayed antibody fragments were selected on Dengue virus particles directly captured from infected Vero cells supernatant by an anti- dengue monoclonal antibody, in order to avoid laborious virus concentration/purification procedures. A total of nine phage-displayed antibody fragments were obtained. Seven of them were highly specific for three of the selector serotypes (two for Dengue 1, four for Dengue 3 and one for Dengue 4). One clone (Dengue 3-selected) cross-reacted with Dengue 1, whereas another (selected with Dengue 2) cross-reacted with the three remaining serotypes. The soluble variants of six antibody fragments recognized their target viruses when used at nanomolar and even subnanomolar concentrations. All phage-displayed antibody fragments were cross-reactive against several strains of distinct genotypes within the corresponding serotype(s). These antibody fragments are potentially useful for the future development of tools for viral diagnosis and serotype identification. The simple phage selection method on captured virus could be applied in a high throughput way to obtain larger panels of antibody fragments to Dengue virus for multiple applications. © 2007 Elsevier B.V. All rights reserved. Keywords: Dengue virus; Phage display; Na¨ ıve antibody library 1. Introduction Among the 70 serologically related viruses of the genus Flavivirus, the Dengue virus complex (four serotypes) is the most important in terms of geographical distribution and exten- sive human morbidity (Monath, 1994). The infection caused by Dengue virus can be characterized by a spectrum of syndromes ranging from mild febrile illness to classical dengue fever and severe and fatal haemorrhagic disease (Gubler, 1998). The lat- ter is often attributed to reinfection by heterologous serotypes ∗ Corresponding author. Tel.: +53 7 2020450; fax: +53 7 2046051. E-mail addresses: sheila@ipk.sld.cu (S. Cabezas), gertrudis.rojas@cigb.edu.cu (G. Rojas), alequis@ipk.sld.cu (A. Pavon), mayling@ipk.sld.cu (M. Alvarez), mpupo@ipk.sld.cu (M. Pupo), gerardo.guillen@cigb.edu.cu (G. Guillen), lupe@ipk.sld.cu (M.G. Guzman). (Halstead and Simasthien, 1970). Dengue infection is thought to induce lifelong immunity against the same virus serotype, but heterotypic immunity is only partial and transient (Sabin, 1952). Therefore, people living in epidemic areas may be susceptible to four infections in their lifetime, and it is not unusual to find more than one serotype co-circulating in an area. Thus, rapid diagnosis and identification of the circulating serotype is crucial for the management of the disease. Mouse monoclonal antibodies (Mabs) obtained through con- ventional hybridoma technology against Dengue virus have proven to be valuable for serotype determination (Henchal et al., 1982) and early diagnosis (Henchal et al., 1983; Xu et al., 2006). Despite hybridoma technology procedures are laborious and time consuming, their use has extended to many labora- tories worldwide and a plethora of useful antibodies has been isolated in this way (Chen et al., 2007; Henchal et al., 1982; Kaufman et al., 1989; Simantini and Banerjee, 1995; Xu et 0166-0934/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2007.09.001