Samuelsen H, Karlsson-Borgå A, Paulsen B S, Wold J K, Rolfsen W
Institute of Pharmacy, University of Oslo, Norway.
Allergy. 1991 Feb;46(2):115-24. doi: 10.1111/j.1398-9995.1991.tb00554.x.
Sera from patients sensitized to Aspergillus fumigatus (A.f.) were screened for specific IgE using sodiumdodecylsulphate-gradient-polyacrylamidegelelectrophoresis (SDSgPAGE) followed by immunoblotting to nitrocellulose. Approximately 25 IgE-binding components were detected. The components of molecular weight 20, 31, 44, 50, 53, 77 and 90 kD were reacting with more than 50% of the patients. The 90, 77 and 20-kD components showed up as the strongest IgE-binding bands. The 20-kD component, called Ag 20 kD, was purified and further characterized. Ag 20 kD was purified to apparent homogeneity. using a combination of size-exclusion chromatography on a Sephacryl S-200 column, preparative isoelectric focusing in a pI 2.5-6.5 gradient, and a Sephadex G-50 Superfine column. Fractions were characterized with protein and carbohydrate analyses, RAST and SDSgPAGE followed by immunoblotting to nitrocellulose. Ag 20 kD was found to be a glycoprotein as it stained with both Coomassie Brilliant Blue and PAS. However, it did not bind Con A, and thus, did probably not contain any terminal alpha-D mannopyranosyl end groups. The relation between mannose, galactose and glucose was found to be 2:1:0.5. The isoelectric point was heterogeneous within pH range 5-6, and the molecular weight was estimated to approximately 20 kD. An increased RAST response was shown for the purified component compared with crude extract using patient sera reacting with Ag 20. The antigen was shown not to be identical with the previously described Ag 3. Neither did it fit the description of Ag 5, 7 or 13 earlier described by the same group. The antigen is going to be used for further immunochemical and clinical investigations, and coupling to other systems for antigen characterization.
使用十二烷基硫酸钠梯度聚丙烯酰胺凝胶电泳（SDSgPAGE），随后进行硝酸纤维素膜免疫印迹，对烟曲霉（A.f.）致敏患者的血清进行特异性IgE筛查。检测到约25种IgE结合成分。分子量为20、31、44、50、53、77和90 kD的成分与超过50%的患者发生反应。90、77和20 kD的成分显示为最强的IgE结合带。对称为Ag 20 kD的20 kD成分进行了纯化并进一步表征。使用Sephacryl S - 200柱上的尺寸排阻色谱、在pI 2.5 - 6.5梯度下的制备性等电聚焦以及Sephadex G - 50 Superfine柱的组合，将Ag 20 kD纯化至表观均一性。通过蛋白质和碳水化合物分析、RAST以及SDSgPAGE随后进行硝酸纤维素膜免疫印迹对各馏分进行表征。发现Ag 20 kD是一种糖蛋白，因为它用考马斯亮蓝和PAS染色均呈阳性。然而，它不结合伴刀豆球蛋白A，因此可能不含有任何末端α - D甘露吡喃糖基端基。发现甘露糖、半乳糖和葡萄糖之间的关系为2:1:0.5。等电点在pH 5 - 6范围内不均一，分子量估计约为20 kD。与使用与Ag 20反应的患者血清的粗提物相比，纯化成分显示出更高的RAST反应。该抗原与先前描述的Ag 3不同。它也不符合同一组先前描述的Ag 5、7或13的描述。该抗原将用于进一步的免疫化学和临床研究，以及与其他系统偶联以进行抗原表征。
