Epitope Binning of Novel Monoclonal Anti F1 and Anti LcrV Antibodies and Their Application in a Simple, Short, HTRF Test for Clinical Plague Detection

Adva Mechaly; Einat B Vitner; Yinon Levy; David Gur; Moria Barlev-Gross; Assa Sittner; Michal Koren; Haim Levy; Emanuelle Mamroud; Morly Fisher

doi:10.3390/pathogens10030285

Epitope Binning of Novel Monoclonal Anti F1 and Anti LcrV Antibodies and Their Application in a Simple, Short, HTRF Test for Clinical Plague Detection

Pathogens. 2021 Mar 2;10(3):285. doi: 10.3390/pathogens10030285.

Authors

Affiliations

¹ The Department of Infectious Diseases, Israel Institute for Biological Research, Ness-Ziona 7410001, Israel.
² The Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona 7410001, Israel.

Abstract

Mouse monoclonal antibodies were raised against plague disease biomarkers: the bacterial capsular protein fraction 1 (F1) and the low-calcium response-LcrV virulence factor (Vag). A novel tandem assay, employing BioLayer Interferometry (BLI), enabled the isolation of antibodies against four different epitopes on Vag. The tandem assay was carried out with hybridoma supernatants, circumventing the need for antibody purification. The BioLayer assay was further adopted for characterization of epitope-repetitive antigens, enabling the discovery of two unique epitopes on F1. The selected antibodies were purified and applied as "oligo-clonal" reagents for the immuno-detection of both biomarkers. The developed Homogenous Time Resolved Fluorescence (HTRF) tests were short (10 min) and simple (no washing steps), allowing for detection of 10 ng/mL F1 and 2.5 ng/mL Vag. The tests were successfully applied for detection of disease biomarkers produced by various Y. pestis strains during growth in blood culture vials.

Keywords: F1; HTRF; LcrV; immuno-detection; monoclonal antibodies; plague.