Preliminary application of monoclonal antibody to Brucella Omp31 in flow cytometry assay / 中华地方病学杂志

ABSTRACT

Objective:Using the monoclonal antibody to Brucella Omp31, flow cytometry (FCM) method for detecting Brucella antigens is established, and to analyze its potential value in clinical diagnosis. Methods:The supernatants of sonicated proteins (SSPs) from Brucella abortus (2308, 104M and S19), Brucella melitensis (M5-90), and Brucella suis (S2) were identified by Western blotting and enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody (mAb) 5H3 to Brucella Omp31, which were prepared by breaking Brucella species with ultra-sonication. The recombinant eukaryotic plasmid (pcDNA3.1-Omp31) was constructed and transfected in 293FT cells, and the expression of Omp31 was detected by Western blotting. THP-1 cells were infected by Brucella melitensis M5-90 strain to simulate mononuclear phagocytes carrying with Brucella spp. To identify the ability of mAb 5H3, FCM for detecting intracellular Brucella was established, mAb 5H3 was labeled with fluorescein isothiocyanate (FITC-5H3) or P-phycoerythrin (PE-5H3), and then the transfected 293FT cells and THP-1 cells invaded by M5-90 strain were individually identified by FCM with FITC-5H3, and sensitivity of FITC-5H3 in FCM was tested. The PBMCs collected from brucellosis patients or normal blood donors were tested by FCM with double mAbs including PE-5H3 and FITC-CD14 to evaluate this method's feasibility in clinical practice. Results:MAb 5H3 was able to identify Brucella melitensis (M5-90) and Brucella suis (S2), as well as Brucella abortus (2308, 104M and S19) with Omp31 gene deletion. The mAb 5H3 labeled with FITC or PE was used for identifying Brucella antigen in various cells by FCM. The results revealed that the proportion of 293FT positive cells expressing Omp31 was about 59.3%, and the proportion of THP-1 positive cells infected by vaccine strain M5-90 was about 6.2%. In addition, the sensitivity of FCM with FITC-5H3 for the 293FT cells transfected with pcDNA3.1-Omp31 was about 4%. The FCM based on double mAbs staining of PE-5H3 and FITC-CD14 was preliminarily established. For brucellosis patients, the proportion of cells (1.93%) stained with the double mAbs in PBMCs was higher than that of normal blood donors (< 0.30%, negative) in FCM. Conclusions:A FCM assay is preliminary established basing on mAb 5H3 against Omp31 for detecting intracellular Brucella. Moreover, we have found that mAb 5H3 could recognize Brucella abortus originally lacking Omp31, which reduces the defect of Omp31 applied in all Brucella species detection. The development of this FCM assay provides a new strategy and usable reagents for brucellosis pathogens diagnosis.