Characterization of novel Omp31 antigenic epitopes of Brucella melitensis by monoclonal antibodies

Abstract

BackgroundBrucellosis is a severe zoonotic disease worldwide. Detection and identification of Brucella species are essential to prevent or treat brucellosis in humans and animals. The outer membrane protein-31 (Omp31) is a major protein of Brucellae except for B. abortus, while the Omp31 antigenic epitopes have not been extensively characterized yet. ResultsA total of 22 monoclonal antibodies (mAbs) were produced against Omp31 of Brucella (B.) melitensis, of which 13 recognized five linear epitopes, 7 reacted with semi-conformational epitopes and 2 reacted with conformational epitopes, respectively. The mAb isotypes were 11 (50%) IgG2a, 5 (23%) IgG1 and 6 (27%) IgM. On the basis of epitope recognition and reactivity levels, 8 mAbs including 3 IgM and 5 IgG clones were considered as highly reactive and potentially diagnostic antibodies. Among these mAbs, 7A3 (IgG1), 5B1 (IgG2a), 2C1 (IgG2a) and 5B3 (IgG2a) reacted with differently conserved linear epitopes of B. melitensis, B. ovis, B. suis and B. canis strains, while 5H3 (IgG2a) highly reacted with a conformational epitope of Omp31 when tested with several immunoassays.ConclusionsThese potent monoclonal antibodies can be used for identifying Omp31 antigens or detecting B. melitensis and other Brucella species beyond B. abortus in vitro or in vivo.

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

“…Recombinant proteins of Omp31, Omp19, Omp16, and periplasmic protein 26 (BP26) were produced from B. melitensis strain in the laboratory (Qiu et al, 2012;He et al, 2016;Li et al, 2017). Omp25 gene (642 bp) from B. melitensis M5-90 genomic DNA was cloned into the pET30a expression vector Yousefi et al, 2016), and designated as pET30a-Omp25.…”

“…BALB/c mice were immunized with purified rOmp25. The hybridoma cells, secreting mAbs to rOmp25, were generated and selected according to a previously reported method (Qiu et al, 2012;Patra et al, 2014;Li et al, 2017). To prepare the ascitis fluid, BALB/c mice sensitized by the liquid paraffin were injected subcutaneously with 10 6 /ml hybridoma cells.…”

“…The infectious recombinant lentivirus (LV-HAGE-Omp25) mediated Omp25 expression in 293FT cell as described previously, mimicking Brucella Omp25 antigen in the infected mammalian cells (Zhang et al, 2012;Li et al, 2017).…”

“…The microwell plate was incubated with a 1:10,000 dilution of goat antimouse IgG and IgM HRP-conjugate antibodies as reported previously (Tiwari et al, 2013;Ahmed et al, 2015). In peptide-ELISA, peptides P1-P10 were used to identify antigenic epitopes recognized by each mAb (Qiu et al, 2012;Li et al, 2017). The double-antibody sandwich ELISA (DAS-ELISA) was used to detect Brucella Omp25 by cross-matching mAbs in pair with a capture mAb coated on the microwell plate and a detection mAb conjugated with HRP (Luo et al, 2012).…”

“…293FT cells were transduced with recombinant lentivirus expressing Omp25 (LV-HAGE-Omp25) at multiplicities of infection (MOI = 5) for 8 h. The transduced 293FT cells were detected by immunofluorescent staining (IFS) with Omp25 mAbs as same as with Omp31 or BP26 described previously (Li et al, 2017). PBMCs were collected from two brucellosis patients (Guangzhou Center for Disease Prevention and Control) and were tested by IFS with mAb 6C12.…”