Journal of Virological Methods 220 (2015) 5–12 Contents lists available at ScienceDirect Journal of Virological Methods j o ur na l ho me pa ge: www.elsevier.com/locate/jviromet Production and characterization of monospecific and bispecific antibodies against dengue virus NS1 protein Advaita Ganguly, Ravindra B. Malabadi, Pravin K. Bhatnagar, Xinli Tang, Dipankar Das, Raimer Loebenberg, Mavanur R. Suresh, Hoon H. Sunwoo ∗ Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 11304-89 Avenue, Edmonton, Alberta T6G 2N8, Canada Article history: Received 25 July 2013 Received in revised form 2 April 2015 Accepted 3 April 2015 Available online 11 April 2015 Keywords: Dengue NS1 Monoclonal antibody Quadroma a b s t r a c t Dengue is a mosquito borne infection, which in recent years has become a major international public health concern. Annually, 100 million dengue virus infections are reported worldwide. The nonstruc- tural protein 1 (NS1) of dengue virus is a useful target for diagnostics of dengue infection since the protein is abundantly circulating in the blood during acute phase of the disease, in both primary and secondary infections. This research paper highlights the development of a panel of Mab and bsMab for dengue NS1 detection. The P148 series of Mabs showed high specificity for recombinant dengue NS1 antigen. These antibodies showed no cross reactivity with recombinant dengue envelope protein and other viral proteins. The hybrid-hybridoma approach to generate the P156.1 and P156.2 bsMabs from the P148 monoclonal antibody method was used during this study. Furthermore, the affinity purification provided good yields of quadromas associated with HRPO in two steps. Direct detection method involved coating of plates with different concentrations of recombinant antigen and detecting with bsMab. Sensi- tive sandwich assay with Mabs and bsMabs was also done. Detection of nonstructural dengue antigens may be of benefit for early and rapid diagnosis of dengue infection due to their long half-life in the blood. © 2015 Published by Elsevier B.V. 1. Introduction Dengue is one of the leading vector borne diseases worldwide, and more than 100 million people are infected every year (Gubler and Meltzer, 1999; Ganguly et al., 2013a). Dengue fever is an important mosquito-borne viral disease of humans. This has been a recurrent phenomenon throughout the tropics in the past decade. During 2002, more than 30 Latin-American countries reported over a million dengue fever (DF) cases with large number of dengue Abbreviations: BSA, bovine serum albumin; bsMab, bispecific monoclonal antibodies; DHF, dengue hemorrhagic fever; ECL, enhanced chemiluminescence; ELISA, enzyme linked immunosorbent assay; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GAM-HRPO, goat anti-mouse horseradish perox- idase; HAT, hypoxanthine, aminopterin, and thymidine; HT, hypoxanthine, aminopterin; HRPO, horseradish peroxidase; IMAC, immobilized metal affin- ity chromatography; Mab, monoclonal antibody; NS1, nonstructural protein 1; OD, optical density; PEG, polyethylene glycol; PBS, phosphate buffered saline; PSG, streptomycin–penicillin–glutamine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TMB, tetramethylbenzidine; TRITC, tetramethylrhodamine isothiocyanate. ∗ Corresponding author at: 3142G KATZ Centre, Faculty of Pharmacy and Phar- maceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8. Tel.: +1 780 492 0247; fax: +1 780 492 1217. E-mail address: hsunwoo@ualberta.ca (H.H. Sunwoo). hemorrhagic fever (DHF) (Whitehorn and Farrar, 2010). Increas- ingly cases of the more severe and potentially lethal DHF and dengue shock syndrome (DSS) are reported with children bearing much of the disease burden (Ganguly et al., 2013b). Dengue virus is endemic in at least 100 countries worldwide and causes more human cases than any other mosquito-borne virus. The mortality rate of DHF in most countries is 5%, primarily among children and young adults. In several Asian countries, this virus is the leading cause of hospitalization and death in children. There have been a large number of dengue epidemics that resulted in enormous economic and human loss in parts of Asia and South America (Whitehorn and Farrar, 2010). Hence, there is an urgent need for diagnostic, prophylactic and therapeutic reagents to manage DHF. Dengue virus has four serotypes, which are antigenically distinct (Calisher et al., 1989; Ganguly et al., 2013c). Among those, the dengue virus nonstructural (NS) 1 protein is a 46–50 kDa glycopro- tein expressed in infected mammalian cells. All 7 NS proteins are intracellular proteins with the exception of dengue NS1 protein, which exists as secreted as well as a membrane-associated protein. Both forms are demonstrated to be immunogenic (Young et al., 2000; Flamand et al., 1999; Falconar and Young, 1990). It was also reported that NS1 is one of 7 NS proteins produced during viral replication. It possesses not only group specific but also type specific determinants and has been recognized as an important http://dx.doi.org/10.1016/j.jviromet.2015.04.004 0166-0934/© 2015 Published by Elsevier B.V.