title: A repertoire of high-affinity monoclonal antibodies specific to S. typhi: as potential candidate for improved typhoid diagnostic.
abstract: 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.
authors.affiliation: Centre for Bio-design and Diagnostics, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad, 121001, Haryana, India.
authors.forename: Chandresh
authors.initials: C
authors.lastname: Sharma
authors.forename: Anurag
authors.initials: A
authors.lastname: Sankhyan
authors.forename: Tarang
authors.initials: T
authors.lastname: Sharma
authors.forename: Naeem
authors.initials: N
authors.lastname: Khan
authors.forename: Susmita
authors.initials: S
authors.lastname: Chaudhuri
authors.forename: Niraj
authors.initials: N
authors.lastname: Kumar
authors.forename: Shinjini
authors.initials: S
authors.lastname: Bhatnagar
authors.forename: Navin
authors.initials: N
authors.lastname: Khanna
authors.forename: Ashutosh
authors.initials: A
authors.lastname: Tiwari
mesh_terms: D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005408:Flagellin / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D012485:Salmonella typhi / Q000276:immunology*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012698:Serologic Tests; D051198:Toll-Like Receptor 5 / Q000276:immunology*; D014435:Typhoid Fever / Q000175:diagnosis* / Q000276:immunology / Q000382:microbiology
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; C495346:TLR5 protein, human; D051198:Toll-Like Receptor 5; D005408:Flagellin