Calera J A, López-Medrano R, Ovejero M C, Puente P, Leal F
Departamento de Microbiología y Genética, Universidad de Salamanca/Consejo Superior de Investigaciones Científicias, Spain.
Infect Immun. 1994 Jun;62(6):2322-33. doi: 10.1128/iai.62.6.2322-2333.1994.
The influence of culture medium and time and temperature of growth on the appearance of Aspergillus nidulans antigens was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining or Western blot (immunoblot), of the proteins present in total cellular extracts or culture supernatants. Samples in the exponential, deceleration, and stationary growth phases were selected by biochemical, morphological, and ultrastructural criteria. Protein and antigen patterns (detected with rabbit antibodies) from total extracts were very similar in all cases, and the major differences observed seemed to depend on the age of the cultures. Culture supernatant patterns were highly dependent on the type of medium (complex or defined) and the age of the culture. Temperature did not significantly influence these results. The reproducible reactivity of selected human sera from aspergilloma-affected individuals was strictly associated with the use of defined media, especially Czapek Dox-AOAC, in both total extracts and culture supernatants. Extended growth times were necessary in the case of metabolic antigens (those obtained from culture supernatants). Screening of a battery of 10 selected human serum samples from patients with aspergilloma or invasive aspergillosis demonstrated that two of the antigens (96 to 98 and 45 kDa) from stationary-phase culture supernatants in Czapek Dox-AOAC medium were consistently reactive. When considered together as one unit, both antigens reacted with more than 50% of the sera, and at least one or the other of the antigens reacted with more than 90% of the sera. Less consistent results were obtained for two somatic antigens (from total cell extracts) of 45 to 50 and 20 to 22 kDa.
通过对全细胞提取物或培养上清液中的蛋白质进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳，随后进行银染或蛋白质印迹法（免疫印迹法），确定了培养基、生长时间和温度对构巢曲霉抗原外观的影响。通过生化、形态学和超微结构标准选择指数生长期、减速期和稳定期的样品。在所有情况下，全提取物的蛋白质和抗原模式（用兔抗体检测）非常相似，观察到的主要差异似乎取决于培养物的年龄。培养上清液模式高度依赖于培养基类型（复合培养基或限定培养基）和培养物的年龄。温度对这些结果没有显著影响。从曲霉肿患者中选择的人血清的可重复反应性与在全提取物和培养上清液中使用限定培养基，特别是察氏 - AOAC培养基密切相关。对于代谢抗原（从培养上清液中获得的抗原），需要延长生长时间。对一组来自曲霉肿或侵袭性曲霉病患者的10份选定人血清样本进行筛选表明，察氏 - AOAC培养基中稳定期培养上清液的两种抗原（96至98 kDa和45 kDa）始终具有反应性。当将这两种抗原作为一个整体考虑时，它们与超过50%的血清发生反应，并且至少其中一种抗原与超过90%的血清发生反应。对于两种45至50 kDa和20至22 kDa的体细胞抗原（来自全细胞提取物），结果不太一致。
