AUTHOR=Yang Xin , He Zuoping , Zhang Guoxia , Lu Jinhui , Zhang Hui , Ren Hui , Tian Yanjun , Yang Heng , Chen Chuangfu , Li Linhai , Fu Yongshui , Allain Jean-Pierre , Li Chengyao , Wang Wenjing 

TITLE=Evaluation of Reactivity of Monoclonal Antibodies Against Omp25 of Brucella spp.

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=10

YEAR=2020

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2020.00145

DOI=10.3389/fcimb.2020.00145

ISSN=2235-2988

ABSTRACT=<p>Brucellosis is a serious zoonosis occurring mainly in developing countries, and its diagnosis is largely dependent on serologic detection and bacterial culture. In this study, we developed the murine monoclonal antibodies (mAbs) against a conserved and major outer membrane protein 25 (Omp25) of <italic>Brucella</italic> species (<italic>B</italic>. spp.) for use in clinical diagnosis. The mAbs to Omp25 were produced by hybridoma technique, which were utilized for developing various immunoassays for detection of <italic>Brucellae</italic>, including Western blot (WB), enzyme-linked immunosorbent assay (ELISA), immunochemical staining (ICS), immunofluorescence staining (IFS), and flow cytometry assay (FCM). A number of five mAbs (2B10, 4A12, 4F10, 6C12, and 8F3) specific to Omp25 were selected, including 2 IgG1, 2 IgG2a, and 1 IgG2b. Among them, mAbs 6C12, 8F3, and 4A12 reacted highly with <italic>B. melitensis</italic> (M5-90), <italic>B. abortus</italic> (S19, 104M, and 2308), and <italic>B. suis strain</italic> (S2). No cross-reactivity with <italic>Yersinia enterocolitica</italic> O:9, <italic>Salmonella</italic> spp., and <italic>Escherichia coli</italic> was found. By mapping Omp25 epitopes, mAb 6C12 was found as reacting with a semi-conformational epitope, and mAbs 4A12 and 8F3 as recognizing a different linear epitope, respectively. The paired mAbs were tested for detecting <italic>Brucella</italic> species, suggesting that 8F3 was suitable for solid phase capture and 6C12 or 4A12 was suitable for conjugation with HRP for detection of <italic>Brucella</italic> Omp25 in ELISA. The FCM was established by mAb 6C12 for detecting intracellular <italic>Brucellae</italic>-infected peripheral blood mononuclear cells (PBMCs) from brucellosis patients. In conclusion, mAbs against Omp25 are precious reagents for detection of <italic>Brucellae</italic> in clinical samples with various immunoassays. mAb 6C12-based FCM could be potentially used for the monitoring of therapeutic efficacy for brucellosis in clinical practice.</p>