Jieun Jang ^1,2 Do Hyung Kwon ^1,2 Ju-Hong Jang ¹ Dong-Gwang Lee ¹ Seo-Hyuk Chang ¹ Min-Young Jeon ¹ Young-Su Jeong ³ Dong-Hyun Song ³ Jeong-Ki Min ¹ Jong-Gil Park ^1,2 Moo-Seung Lee ¹ Baek-Soo Han ¹ Wonjun Yang ^1* Nam-Kyung Lee ^1* Jangwook Lee ^1,2*

• ¹ Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea
• ² Korea University of Science and Technology, Daejeon, Republic of Korea
• ³ Agency for Defense Development, Daejeon, Republic of Korea

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.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.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 × 10-10 M and 1.32 × 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 from 0.062 ng/mL in PBS to 0.064 ng/mL in skim milk matrices.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..