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- Title
Development of a novel sandwich immunoassay based on targeting recombinant Francisella outer membrane protein A for the diagnosis of tularemia.
- Authors
Jieun Jang; Do Hyung Kwon; Ju-Hong Jang; Dong-Gwang Lee; Seo-Hyuk Chang; Min-Young Jeon; Young-Su Jeong; Dong-Hyun Song; Jeong-Ki Min; Jong-Gil Park; Moo-Seung Lee; Baek-Soo Han; Wonjun Yang; Nam-Kyung Lee; Jangwook Lee
- Abstract
Introduction: Tularemia, caused by the bacterium Francisella tularensis, poses health risks to humans and can spread through a variety of routes. It has also been classified as a Tier 1 Select agent by the CDC, highlighting its potential as a bioterrorism agent. Moreover, it is difficult to diagnose in a timely fashion, owing to the non-specific nature of tularemia infections. Rapid, sensitive, and accurate detection methods are required to reduce mortality rates. We aimed to develop antibodies directed against the outer membrane protein A of F. tularensis (FopA) for rapid and accurate diagnosis of tularemia. Methods: We used a baculovirus insect cell expression vector system to produce the FopA antigen and generate anti-FopA antibodies through immunization of BALB/c mice. We then employed hybridoma and phage display technologies to screen for antibodies that could recognize unique epitopes on FopA. Result: Two monoclonal antibodies, 6B12 and 3C1, identified through phage display screening specifically bound to recombinant FopA in a dose-dependent manner. The binding affinity of the anti-FopA 6B12 and 3C1 antibodies was observed to have an equilibrium dissociation constant of 1.76 x 10-10 M and 1.32 x 10-9 M, respectively. These antibodies were used to develop a sandwich ELISA system for the diagnosis of tularemia. This assay was found to be highly specific and sensitive, with detection limits ranging from0.062 ng/mL in PBS to 0.064 ng/mL in skim milk matrices. Discussion: Our findings demonstrate the feasibility of a novel diagnostic approach for detecting F. tularensis based on targeting FopA, as opposed to existing tests that target the bacterial lipopolysaccharide.
- Subjects
FRANCISELLA tularensis; TULAREMIA; MEMBRANE proteins; SKIM milk; DETECTION limit
- Publication
Frontiers in Cellular & Infection Microbiology, 2024, p01
- ISSN
2235-2988
- Publication type
Article
- DOI
10.3389/fcimb.2024.1455259