IMMUNOLOGICAL STUDIES OF T. CRUZI METACYCLIC STAGES

恰加斯病在亚环类侵入和感染后加重 克氏锥虫的锥鞭毛体（MC）。 免疫小鼠 辐射减毒的MC抵抗毒性MC的感染。 的融合体 来自具有骨髓瘤的这种免疫小鼠的B细胞产生杂交瘤， 克隆后，产生与阶段反应的单克隆抗体（MAbs） 和种特异性抗原（Ags），中和MC，或者当被动 转移保护收件人。 MAb将从该面板中选择， 它们能够中和MCs并与表位复合的基础 不同的MC Ag，如通过蛋白质印迹分析（WBA）所示。 抗独特型 将制备针对每种MAb的抗体（AidAb）。 单克隆抗体免疫亲和性 层析将用于分离MC Ag。 的均一性和 每种Ag的分子量将通过十二烷基钠 硫酸盐-聚丙烯酰胺凝胶电泳和WBA。 蛋白质和 每种Ag的多糖组成将与其作用相关 酶和化学处理对血清学反应性的影响。 将在用每种疫苗免疫的小鼠中监测寄生虫血症和死亡率。 MCs攻毒感染后的MC Ag或AidAb。 小鼠接受 被动转移的单克隆抗体，类似地接种MC，将被 评估以比较它们对感染的抵抗力， 免疫组。 单克隆抗体介导的抗原与宿主细胞的相互作用 将在用每种AidAb处理的培养巨噬细胞中检查受体 或正常的γ球蛋白。 巨噬细胞也将暴露于经治疗的MC 单克隆抗体或正常球蛋白的抗体。 在孵育间隔后， 洗涤后，计数贴壁MC和感染细胞的数量 为了确定AidAb是否阻断巨噬细胞表面或MAb上的受体， 影响MC附着或内化。 AidAb亲和层析 分离的受体将被生物化学地定义，并且MC Ag和 受体将通过间接荧光Ab显微术定位。 来自Mc-immunized和Mc-infected小鼠的抗血清和来自人的抗血清， 将在血清学试验中比较急性和慢性恰加斯病 与每个MC Ag和AidAb，以确定其反应性和时间 Ab合成的关系。 这项研究将定义疫苗 MC Ag和AidAb的潜力，并可提供敏感和特异性 用于人类感染的血清诊断试剂。

Chagas' disease ensues after invasion and infection by metacyclic trypomastigotes (MCs) of Trypanosoma cruzi. Mice immunized with irradiation-attenuated MCs resist infections by virulent MCs. Fusions of B-cells from such immunized mice with myelomas generate hybridomas which after cloning, produce monoclonal antibodies (MAbs) that react with stage- and species-specific antigens (Ags), neutralize MCs, or may when passively transferred protect recipients. MAbs will be selected from this panel on the basis of their abilities to neutralize MCs and complex with epitopes on different MC Ags as shown by Western blot analyses (WBA). Antiidiotype antibodies (AidAbs) will be prepared against each MAb. MAb-immunoaffinity chromatography will be used to isolate MC Ags. The homogeneity and molecular weight of each Ag will be assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoroesis and WBA. The protein and polysaccharide composition of each Ag will be correlated with the effects of enzymatic and chemical treatments on serological reactivity. Parasitemias and mortalities will be monitored in mice immunized with each MC Ag or AidAb after challenge infections with MCs. Mice receiving passively transferred MAbs, similarly inoculated with MCs, will be evaluated to compare their resistance to infections with the actively immunized groups. Interactions between MAb-defined Ags and host cell receptors will be examined in cultured macrophages treated with each AidAb or normal gamma globulins. Macrophages will also be exposed to MCs treated with each MAb or the normal globulins. After intervals of incubation and washing, the numbers of adherent MCs and infected cells will be enumerated to determine whether AidAbs block receptors on macrophage surfaces or MAbs affect MC attachment or internalization. AidAb affinity chromatography isolated receptors will be defined biochemically and both the MC Ags and receptors will be localized by indirect fluorescent Ab microscopy. Antisera from Mc-immunized and -infected mice and antisera from humans with a cute and chronic Chagas' disease will be compared in serological tests with each MC Ag and AidAb to establish their reactivities and the temporal relationships of Ab synthesis. The research will define the vaccine potential of the MC Ags and AidAbs and may provide sensitive and specific reagents for the serodiagnosis of infections in humans.