Reiss E, Huppert M, Cherniak R
Curr Top Med Mycol. 1985;1:172-207. doi: 10.1007/978-1-4613-9547-8_7.
Antigens in coccidioidin were compared with purified subfractions via tandem immunoelectrophoresis (IEP) and by a combination of advancing line and crossed IEP. Rocket IEP was suitable for titrating the reactions and showing the relationship between column fractions. These techniques required multicomponent antisera produced by hyperimmunization over many months and by the use of known standard migration pairs. The IEP variations were used to chart the development of antisera against coccidioidin factors, to monitor antigen purifications, and to test the immunochemical homogeneity of an isolated antigen. Mannose-based heteroglycans of Cryptococcus neoformans were recovered from the culture filtrate. After precipitation of the major viscous glucuronoxylomannan (GXM) with ethanol or cetyltrimethylammonium bromide, the supernate is reserved because it contains a galactoxylomannan (GalXM). After removal of glucuronic acid from the GXM, the resulting xylomannan of serotype A was amenable to 13C-nuclear magnetic resonance (NMR) spectrometry; it revealed nonreducing xylose, alpha-1,3-mannose, and alpha-1,2/1,3 disubstituted mannose, thus confirming by an independent means what was previously known. The characterization sequence of GalXM included: (1) gas-liquid chromatography (GLC) of neutral sugars as peracetylated aldononitriles; (2) methylation-fragmentation GLC mass spectrometry to determine the glycosidic linkages; and (3) 13C-NMR showing similarities to mannan of Saccharomyces cerevisiae. Affinity chromatography of the GalXM on concanavalin A separated the galactoxylo component from an adsorbed mannoprotein. Selection of monoclonal antibodies (MAbs) relies on presumptive enzyme immunoassays (EIAs) or radioimmunoassays for rapid screening of clones and for determination of isotypes; however, higher resolution confirmatory tests are needed to obtain MAbs of desired specificity. MAbs against Candida tropicalis mannan were labeled with horseradish peroxidase to use for detecting mannan in serum. MAbs against the partially purified "m" factor of histoplasmin were characterized by the enzyme-linked immunoelectro-transfer blot technique (EITB), revealing unsuspected complexity in the antigen. Secreted proteins of Nocardia asteroides were isoelectrically focused; three proteins, identified by EITB as promising to be specific for that actinomycete, were cut out of gels and used to immunize mice for production of MAbs. The fimbriae of Actinomyces viscosus and A. naeslundii that mediate lactose-reversible coagglutination with Streptococcus sanguis have been used to evoke MAbs.(ABSTRACT TRUNCATED AT 400 WORDS)
通过串联免疫电泳（IEP）以及前进线免疫电泳和交叉免疫电泳相结合的方法，对球孢子菌素中的抗原与纯化亚组分进行了比较。火箭免疫电泳适用于滴定反应并显示柱级分之间的关系。这些技术需要通过数月的超免疫以及使用已知的标准迁移对产生的多组分抗血清。免疫电泳变异被用于绘制针对球孢子菌素因子的抗血清的发展过程、监测抗原纯化以及测试分离抗原的免疫化学均一性。新型隐球菌的基于甘露糖的杂聚糖是从培养滤液中回收的。用乙醇或十六烷基三甲基溴化铵沉淀主要的粘性葡糖醛酸木聚糖甘露聚糖（GXM）后，保留上清液，因为其中含有半乳甘露聚糖（GalXM）。从GXM中去除葡糖醛酸后，所得的A型木聚糖甘露聚糖适合进行13C核磁共振（NMR）光谱分析；它揭示了非还原木糖、α-1,3-甘露糖和α-1,2/1,3双取代甘露糖，从而通过一种独立的方法证实了先前已知的情况。GalXM的表征序列包括：（1）将中性糖作为全乙酰化醛糖腈进行气液色谱（GLC）分析；（2）甲基化-碎片化GLC质谱分析以确定糖苷键；（3）13C-NMR显示与酿酒酵母甘露聚糖相似。GalXM在伴刀豆球蛋白A上的亲和色谱分离出了吸附的甘露糖蛋白中的半乳甘露糖组分。单克隆抗体（MAb）的筛选依赖于推测性酶免疫测定（EIA）或放射免疫测定，用于快速筛选克隆以及确定同种型；然而，需要更高分辨率的确认性试验来获得具有所需特异性的MAb。针对热带念珠菌甘露聚糖的MAb用辣根过氧化物酶标记，用于检测血清中的甘露聚糖。针对组织胞浆菌素部分纯化的“m”因子的MAb通过酶联免疫电转移印迹技术（EITB）进行表征，揭示了该抗原中未被怀疑的复杂性。星形诺卡菌的分泌蛋白进行了等电聚焦；通过EITB鉴定为有望对该放线菌具有特异性的三种蛋白质从凝胶中切出，用于免疫小鼠以产生MAb。粘性放线菌和内氏放线菌的菌毛介导与血链球菌的乳糖可逆性协同凝集，已被用于引发MAb。（摘要截短至400字）
