Center of Edaphology and Applied Biology of the Segura (CEBAS)-CSIC, Department of Stress Biology and Plant Pathology, PO Box 164, 30100, Espinardo, Murcia, Spain.
Abiopep S.L., R&D Department, Parque Científico de Murcia, Ctra. de Madrid, Km 388, Complejo de Espinardo, Edf. R, 2º, 30100, Espinardo, Murcia, Spain.
J Virol Methods. 2020 Apr;278:113837. doi: 10.1016/j.jviromet.2020.113837. Epub 2020 Feb 21.
Melon necrotic spot virus (MNSV) is endemic in cucurbit crops worldwide, causing epidemic outbreaks from time to time. MNSV is transmitted in nature by a soil-inhabiting fungus and also through seeds, making its detection in seed certification programs a necessity. Polyclonal antisera and RT-PCR-based detection assays have been developed for MNSV, but up to now no monoclonal antibodies (mAbs) have been described for this virus. In this study, we have produced mAbs in BALB/c mice against the MNSV over-expressed coat protein (CP). Titers of the antibodies produced against the recombinant MNSV CP ranged around 10-10 and the IgG yields for each mAb from ascitic fluids ranged from 1.51 to 6 mg/mL. Supernatants from ten hybridoma cell lines were evaluated in Western blot analysis and seven of them efficiently recognized the MNSV CP in crude extracts of MNSV-infected leaf material; the 2D4H4 hybridoma cell line was selected for further purification and characterization. The isotype of the 2D4H4 immunoglobulin class was identified as IgG2a and kappa light-chain. Western-blot analyses showed that mAb 2D4H4 provided sensitive and specific detection of MNSV. A TAS-ELISA protocol was developed for mAb 2D4H4. Using this protocol, limits of detection of 1:20,480 and 1:10,240 (g/mL, w/v) were attained for the homologous isolate and a heterologous MNSV isolate, respectively. Moreover, mAb 2D4H4 was used successfully to localize the MNSV CP in infected cells by immunocytochemistry/transmission electron microscopy, illustrating the usefulness of this mAb for advanced cellular studies.
甜瓜坏死斑点病毒（MNSV）在世界范围内的葫芦科作物中流行，不时引发疫情爆发。MNSV 在自然界中通过土壤中栖息的真菌传播，也通过种子传播，因此在种子认证计划中需要对其进行检测。已经针对 MNSV 开发了多克隆抗血清和基于 RT-PCR 的检测方法，但迄今为止，尚未有针对该病毒的单克隆抗体（mAb）。在这项研究中，我们使用 BALB/c 小鼠针对过表达的 MNSV 外壳蛋白（CP）产生了 mAb。针对重组 MNSV CP 产生的抗体滴度约为 10-10，从腹水液中获得的每种 mAb 的 IgG 产量范围为 1.51 至 6 mg/mL。在 Western blot 分析中评估了来自十个杂交瘤细胞系的上清液，其中七个在 MNSV 感染叶片材料的粗提物中有效地识别了 MNSV CP；选择 2D4H4 杂交瘤细胞系进行进一步的纯化和表征。2D4H4 免疫球蛋白类别的同型鉴定为 IgG2a 和 kappa 轻链。Western blot 分析表明，mAb 2D4H4 提供了对 MNSV 的敏感和特异性检测。为 mAb 2D4H4 开发了 TAS-ELISA 方案。使用该方案，对同源分离株和异源 MNSV 分离株的检测限分别达到了 1:20,480 和 1:10,240（g/mL，w/v）。此外，mAb 2D4H4 成功地用于通过免疫细胞化学/透射电子显微镜定位感染细胞中的 MNSV CP，说明了该 mAb 用于高级细胞研究的有用性。
