A repertoire of high-affinity monoclonal antibodies specific to S. typhi: as potential candidate for improved typhoid diagnostic
- 198 Downloads
Typhoid fever is a significant global health problem with highest burden on the developing world. The severity of typhoid is often underestimated, and currently available serological diagnostic assays are inadequate due to lack in requisite sensitivity and specificity. This underlines an absolute need to develop a reliable and accurate diagnostics that would benefit long-term disease control and treatment and to understand the real disease burden. Here, we have utilized flagellin protein of S. typhi that is surface accessible, abundantly expressed, and highly immunogenic, for developing immunodiagnostic tests. Flagellin monomers are composed of conserved amino-terminal and carboxy-terminal, and serovar-specific middle region. We have generated a panel of murine monoclonal antibodies (mAbs) against the middle region of flagellin, purified from large culture of S. typhi to ensure its native conformation. These mAbs showed unique specificity and very high affinity toward S. typhi flagellin without showing any cross-reactivity with other serovars. Genetic analysis of mAbs also revealed high frequency of somatic mutation due to antigenic selection process across variable region to achieve high binding affinity. These antibodies also displayed stable binding in stringent reaction conditions for antigen–antibody interactions, like DMSO, urea, KSCN, guanidinium HCl, and extremes of pH. One of the mAbs potentially reversed the TLR5-mediated immune response, in vitro by inhibiting TLR5–flagellin interaction. In our study, binding of these mAbs to flagellin, with high affinity, present on bacterial surface, as well as in soluble form, validates their potential use in developing improved diagnostics with significantly higher sensitivity and specificity.
KeywordsFlagellin S.typhi Typhoid fever Monoclonal antibodies TLR5 Typhoid diagnostics
The authors gratefully acknowledge Dr. Ayub Qadri (National Institute of Immunology, New Delhi, India) and Dr. Bhabatosh Das (Translational Health Science and Technology Institute), for providing Salmonella strains. This study was supported by grant from Department of Biotechnology, Government of India, to Centre for Bio-design and Diagnostics, Translational Health Science and Technology Institute, and the core grant of Translational Health Science and Technology Institute to AT. C S was supported by Innovation Award from Centre for Bio-design and Diagnostics.
Conflict of interest
The authors declare that they have no conflict of interest.
All procedures performed in this study involving animals were in accordance with the ethical standards of the institution or practice at the International Centre for Genetic Engineering and Biotechnology, New Delhi, India.
- 13.World Health Organization. Background document: the diagnosis, treatment and prevention of typhoid fever. World Health Organization; 2003. http://www.who.int/immunization/documents/WHO_VB_03.07/en/. Accessed 06 Dec 2012.
- 15.Widal F. Serodiagnostique de la fievre typhoid. Semaine Med. 1896;16:259.Google Scholar
- 21.Preechakasedkit P, Pinwattana K, Dungchai W, Siangproh W, Chaicumpa W, Tongtawe P, et al. Development of a one-step immunochromatographic strip test using gold nanoparticles for the rapid detection of Salmonella typhi in human serum. Biosens Bioelectron. 2012;31(1):562–6. doi: 10.1016/j.bios.2011.10.031.PubMedCrossRefGoogle Scholar
- 28.de Vries N, Zwaagstra KA, Huis in’t Veld JH, van Knapen F, van Zijderveld FG, Kusters JG. Production of monoclonal antibodies specific for the i and 1,2 flagellar antigens of Salmonella typhimurium and characterization of their respective epitopes. Appl Environ Microbiol. 1998;64(12):5033–8.PubMedCentralPubMedGoogle Scholar
- 56.Smith MF Jr, Mitchell A, Li G, Ding S, Fitzmaurice AM, Ryan K, et al. Toll-like receptor (TLR) 2 and TLR5, but not TLR4, are required for Helicobacter pylori-induced NF-kappa B activation and chemokine expression by epithelial cells. J Biol Chem. 2003;278(35):32552–60. doi: 10.1074/jbc.M305536200.PubMedCrossRefGoogle Scholar
- 59.Stecher B, Hapfelmeier S, Muller C, Kremer M, Stallmach T, Hardt WD. Flagella and chemotaxis are required for efficient induction of Salmonella enterica serovar Typhimurium colitis in streptomycin-pretreated mice. Infect Immun. 2004;72(7):4138–50. doi: 10.1128/IAI.72.7.4138-4150.2004.PubMedCentralPubMedCrossRefGoogle Scholar