DataSheet_1_Monoclonal antibody-based localization of major diagnostic antigens in metacestode tissue, excretory/secretory products, and extracellular vesicles of Echinococcus species.docx

Alveolar (AE) and cystic echinococcosis (CE) are severe parasitic zoonoses caused by the larval stages of Echinococcus multilocularis and E. granulosus sensu lato, respectively. A panel of 7 monoclonal antibodies (mAbs) was selected against major diagnostic epitopes of both species. The binding capacity of the mAbs to Echinococcus spp. excretory/secretory products (ESP) was analyzed by sandwich-ELISA, where mAb Em2G11 and mAb EmG3 detected in vitro extravesicular ESP of both E. multilocularis and E. granulosus s.s. These findings were subsequently confirmed by the detection of circulating ESP in a subset of serum samples from infected hosts including humans. Extracellular vesicles (EVs) were purified, and the binding to mAbs was analyzed by sandwich-ELISA. Transmission electron microscopy (TEM) was used to confirm the binding of mAb EmG3 to EVs from intravesicular fluid of Echinococcus spp. vesicles. The specificity of the mAbs in ELISA corresponded to the immunohistochemical staining (IHC-S) patterns performed on human AE and CE liver sections. Antigenic small particles designated as ‘‘spems’’ for E. multilocularis and ‘‘spegs’’ for E. granulosus s.l. were stained by the mAb EmG3IgM, mAb EmG3IgG1, mAb AgB, and mAb 2B2, while mAb Em2G11 reacted with spems and mAb Eg2 with spegs only. The laminated layer (LL) of both species was strongly visualized by using mAb EmG3IgM, mAb EmG3IgG1, mAb AgB, and mAb 2B2. The LL was specifically stained by mAb Em2G11 in E. multilocularis and by mAb Eg2 in E. granulosus s.l. In the germinal layer (GL), including the protoscoleces, a wide staining pattern with all structures of both species was observed with mAb EmG3IgG1, mAb EmG3IgM, mAb AgB, mAb 2B2, and mAb Em18. In the GL and protoscoleces, the mAb Eg2 displayed a strong E. granulosus s.l. specific binding, while mAb Em2G11 exhibited a weak granular E. multilocularis specific reaction. The most notable staining pattern in IHC-S was found with mAb Em18, which solely bound to the GL and protoscoleces of Echinococcus species and potentially to primary cells. To conclude, mAbs represent valuable tools for the visualization of major antigens in the most important Echinococcus species, as well as providing insights into parasite-host interactions and pathogenesis.

肺泡型包虫病（AE）和囊型包虫病（CE）是由多房棘球绦虫的幼虫阶段和广义上的棘球绦虫E. granulosus引起的严重的人畜共患病。针对两种物种的主要诊断表位，选出了7种单克隆抗体（mAbs）。通过夹心ELISA分析了这些mAbs对棘球绦虫属排泄/分泌产物（ESP）的结合能力，其中mAb Em2G11和mAb EmG3在体外检测到多房棘球绦虫和广义棘球绦虫s.s.的囊外ESP。这些发现随后通过检测感染宿主（包括人类）血清样本中的循环ESP得到了证实。外泌体（EVs）被纯化，并通过夹心ELISA分析了其与mAbs的结合。透射电子显微镜（TEM）被用于确认mAb EmG3与棘球绦虫属囊泡内液EVs的结合。在ELISA中，mAbs的特异性与对人类AE和CE肝脏切片进行的免疫组化染色（IHC-S）模式相对应。标记为“spems”的抗原性小颗粒用于多房棘球绦虫，而“spegs”用于广义棘球绦虫s.l.，这些颗粒由mAb EmG3IgM、mAb EmG3IgG1、mAb AgB和mAb 2B2染色，而mAb Em2G11仅与spems反应，mAb Eg2仅与spegs反应。使用mAb EmG3IgM、mAb EmG3IgG1、mAb AgB和mAb 2B2，两种物种的层状层（LL）被强烈可视化。在棘球绦虫属的LL中，mAb Em2G11特异地进行了染色，而在广义棘球绦虫s.l.中，mAb Eg2进行了染色。在生发层（GL），包括原头节，mAb EmG3IgG1、mAb EmG3IgM、mAb AgB、mAb 2B2和mAb Em18观察到广泛的染色模式，涉及两种物种的所有结构。在GL和原头节中，mAb Eg2表现出对广义棘球绦虫s.l.的强特异性结合，而mAb Em2G11表现出对多房棘球绦虫的弱颗粒特异性反应。在IHC-S中，最显著的染色模式由mAb Em18产生，它仅与棘球绦虫属的GL和原头节以及可能的原发性细胞结合。总之，mAbs是可视化最重要的棘球绦虫属中主要抗原的有价值工具，同时也为寄生虫-宿主相互作用和发病机制提供了见解。