Abstract
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 Brucella species (B. 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 Brucellae, 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 B. melitensis (M5-90), B. abortus (S19, 104M, and 2308), and B. suis strain (S2). No cross-reactivity with Yersinia enterocolitica O:9, Salmonella spp., and Escherichia coli 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 Brucella species, suggesting that 8F3 was suitable for solid phase capture and 6C12 or 4A12 was suitable for conjugation with HRP for detection of Brucella Omp25 in ELISA. The FCM was established by mAb 6C12 for detecting intracellular Brucellae-infected peripheral blood mononuclear cells (PBMCs) from brucellosis patients. In conclusion, mAbs against Omp25 are precious reagents for detection of Brucellae 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.
Highlights
Brucellosis is a severe zoonotic disease distributed worldwide, especially in the developing world (Wang et al, 2015)
Western blot analysis showed all five monoclonal antibodies (mAbs) were highly reactive with denatured Recombinant Omp25 (rOmp25), while among them three mAbs (6C12, 8F3, and 4A12) strongly reacted with the denatured native membrane protein extracts (NMPs) of B. melitensis and two (2B10 and 4F10) reacted weakly (Figure 1C)
One patient (No 8) had 1.13% of stained monocytes with unclear boundary by flow cytometry assay (FCM). These results suggested that FCM with mAb 6C12 might be a practical assay to determine the frequency of intracellular Brucellae-infected peripheral blood mononuclear cells (PBMCs) in individual brucellosis patients with clinical diagnosis
Summary
Brucellosis is a severe zoonotic disease distributed worldwide, especially in the developing world (Wang et al, 2015). Goats, cattle, and pigs infected by Brucella species are the main sources of human brucellosis (Ducrotoy et al, 2018). Diagnosis of brucellosis mainly depends on serological methods used to detect antibodies against Brucellae in infected animals or humans (Araj, 2010). Omp is highly conserved in various types and subtypes of Brucellae and induces a strong immune response (Cloeckaert et al, 2002; Goel et al, 2013; Ma et al, 2015). It might be a useful diagnostic target for brucellosis. We aimed to generate novel monoclonal antibodies (mAbs) to Omp and to develop new immunoassays for diagnosis of brucellosis or evaluation of therapeutic efficacy of brucellosis in clinical practice
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