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

doi:10.3390/pathogens10030285

Adva Mechaly, Moria Barlev-Gross + Show 8 more

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https://doi.org/10.3390/pathogens10030285

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Journal: Pathogens	Publication Date: Mar 2, 2021
Citations: 2	License type: CC BY 4.0

Affiliation: Israel Institute for Biological Research

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.

Highlights

IntroductionThe growing interest in rapid diagnostic tools for medical use (e.g., clinical and point-of-care diagnostics) has encouraged the development of fast, simple-to-operate immunoassays
The growing interest in rapid diagnostic tools for medical use has encouraged the development of fast, simple-to-operate immunoassays
Monoclonal antibodies against the recombinant Yersinia pestis antigens fraction 1 (F1) and Vag were generated and screened by either the conventional ELISA-screening method or by a flow cytometry-based screening method [23]

Summary

Introduction

The growing interest in rapid diagnostic tools for medical use (e.g., clinical and point-of-care diagnostics) has encouraged the development of fast, simple-to-operate immunoassays. One such assay is the Homogeneous Time Resolved Fluorescent (HTRF). The efficiency of the donor-acceptor energy transfer is inversely proportional to the sixth power of the distance between both molecules [3]. Both fluorophores have to be in close spatial proximity (

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