Department of Transfusion Medicine, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China.
The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China.
Front Cell Infect Microbiol. 2020 Apr 21;10:145. doi: 10.3389/fcimb.2020.00145. eCollection 2020.
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 species (. 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 , 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 (M5-90), (S19, 104M, and 2308), and (S2). No cross-reactivity with O:9, spp., and 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 species, suggesting that 8F3 was suitable for solid phase capture and 6C12 or 4A12 was suitable for conjugation with HRP for detection of Omp25 in ELISA. The FCM was established by mAb 6C12 for detecting intracellular -infected peripheral blood mononuclear cells (PBMCs) from brucellosis patients. In conclusion, mAbs against Omp25 are precious reagents for detection of 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.
布鲁氏菌病是一种主要发生在发展中国家的严重人畜共患病，其诊断在很大程度上依赖于血清学检测和细菌培养。在本研究中，我们针对种的主要外膜蛋白 25（Omp25）开发了针对的单克隆抗体（mAbs），用于临床诊断。Omp25 的 mAbs 是通过杂交瘤技术产生的，用于开发各种检测的免疫分析，包括 Western blot（WB）、酶联免疫吸附试验（ELISA）、免疫化学染色（ICS）、免疫荧光染色（IFS）和流式细胞术检测（FCM）。选择了针对 Omp25 的 5 种 mAbs（2B10、4A12、4F10、6C12 和 8F3），包括 2 种 IgG1、2 种 IgG2a 和 1 种 IgG2b。其中，mAbs 6C12、8F3 和 4A12 与（M5-90）、（S19、104M 和 2308）和（S2）高度反应。与 0:9、种和 无交叉反应。通过映射 Omp25 表位，发现 mAb 6C12 与半构象表位反应，mAbs 4A12 和 8F3 分别识别不同的线性表位。配对的 mAbs 用于检测种，表明 8F3 适合固相捕获，6C12 或 4A12 适合与 HRP 缀合用于 ELISA 中检测 Omp25。FCM 通过 mAb 6C12 建立，用于检测布鲁氏菌病患者外周血单个核细胞（PBMCs）中的细胞内感染。总之，针对 Omp25 的 mAbs 是各种免疫分析检测临床样本中 的宝贵试剂。基于 mAb 6C12 的 FCM 可潜在用于临床实践中布鲁氏菌病治疗效果的监测。
