Laboratorio de Parasitología, Universidad San Pablo CEU, Madrid, Spain.
Centers for Disease Control and Prevention, Atlanta, GA, USA.
Parasit Vectors. 2017 Nov 9;10(1):560. doi: 10.1186/s13071-017-2503-z.
Microsporidia are intracellular obligate parasites traditionally associated with immunosuppressed patients; their detection in immunocompetent patients has increased, highlighting their possible importance as emerging pathogens. Detection of spores in stools, urine, body fluids and tissues is difficult and immunological techniques such as immunofluorescence have proved to be a useful and reliable tool in the diagnosis of human microsporidiosis. For this reason, we have produced and characterized monoclonal antibodies (MAbs) specific for Encephalitozoon intestinalis (the second most frequent microsporidian infecting humans), and other Encephalitozoon species, that can be used in different diagnostic techniques.
Seven MAbs were selected in accordance with their optical density (OD). Four (4C4, 2C2, 2E5 and 2H2) were isotype IgG2a; two (3A5 and 3C9) isotype IgG3, and one Mab, 1D7, IgM isotype. The selected monoclonal antibody-secreting hybridomas were characterized by indirect immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), Western blot, immunoelectron microscopy (Immunogold) and in vitro cultures. The study by IFAT showed different behavior depending on the MAbs studied. The MAbs 4C4, 2C2, 2E5 and 2H2 showed reactivity against epitopes in the wall of the spore (exospore and endospore) epitopes located in Encephalitozoon sp. spores, whereas the MAbs 3A5, 1D7 and 3C9 showed reactivity against internal epitopes (cytoplasmic contents or sporoplasm) of E. intestinalis spores. All MAbs recognized the developing parasites in the in vitro cultures of E. intestinalis. Additionally, 59 formalin-fixed stool samples that had been previously analyzed were screened, with 26 (44%) presenting microsporidian spores (18 samples with E. intestinalis and 8 samples with Enterocytozoon bieneusi). Detection of microsporidian spores by microscopy was performed using Calcofluor stain, Modified Trichrome, Quick-Hot Gram Chromotrope, as well as IFAT using MAbs 4C4, 2C2, 2E5 and 2H2. The 4 MAbs tested clearly recognized the larger spores corresponding to E. intestinalis, but showed no reactivity with Enterocytozoon bieneusi spores. The mass spectrometry and proteomic study revealed that the Mabs 4C4, 2C2, 2E5 and 2H2 recognized the Spore Wall Protein 1 (SWP1) as the antigenic target.
The IFAT-positive MAbs exhibited excellent reactivity against spores and developmental stages, permitting their use in human and animal diagnosis. The epitopes recognized (exospore, endospore and cytoplasmic contents) by the different MAbs developed need further study, and may reveal potential targets for vaccine development, immunotherapy and chemotherapy.
微孢子虫是传统上与免疫抑制患者相关的细胞内专性寄生虫；它们在免疫功能正常的患者中的检测增加，突显了它们作为新兴病原体的可能重要性。在粪便、尿液、体液和组织中检测孢子较为困难，免疫荧光等免疫学技术已被证明是诊断人类微孢子虫病的有用且可靠的工具。为此，我们生产并鉴定了针对肠微孢子虫（感染人类的第二大常见微孢子虫）和其他肠微孢子虫的特异性单克隆抗体（MAbs），可用于不同的诊断技术。
根据光密度（OD）选择了 7 种 MAbs。其中 4 种（4C4、2C2、2E5 和 2H2）为 IgG2a 同种型；2 种（3A5 和 3C9）为 IgG3 同种型，而一种 Mab，1D7，为 IgM 同种型。通过间接免疫荧光抗体试验（IFAT）、酶联免疫吸附试验（ELISA）、Western blot、免疫电镜（免疫金）和体外培养对所选分泌单克隆抗体的杂交瘤进行了鉴定。IFAT 研究表明，不同的 MAb 表现出不同的行为。MAbs 4C4、2C2、2E5 和 2H2 针对孢子壁（外孢子和内孢子）表位显示出反应性，这些表位位于肠微孢子虫属孢子中，而 MAb 3A5、1D7 和 3C9 则针对肠微孢子虫属孢子的内部表位（细胞质内容物或孢子质）显示出反应性。所有 MAb 均识别体外培养的肠微孢子虫中的发育寄生虫。此外，对之前分析过的 59 份福尔马林固定粪便样本进行了筛选，其中 26 份（44%）显示有微孢子虫孢子（18 份为肠微孢子虫，8 份为肠内原虫）。使用 Calcofluor 染色、改良三色、Quick-Hot Gram Chromotrope 以及使用 MAb 4C4、2C2、2E5 和 2H2 的 IFAT 对微孢子虫孢子进行显微镜检测。测试的 4 种 MAb 清楚地识别出较大的肠微孢子虫孢子，但对 Enterocytozoon bieneusi 孢子无反应。质谱和蛋白质组学研究表明，MAbs 4C4、2C2、2E5 和 2H2 识别出孢子壁蛋白 1（SWP1）作为抗原靶标。
IFA 阳性的 MAb 对孢子和发育阶段表现出优异的反应性，允许其用于人类和动物诊断。不同 MAb 识别的表位（外孢子、内孢子和细胞质内容物）需要进一步研究，这可能揭示出疫苗开发、免疫疗法和化学疗法的潜在靶点。
