Iacovacci P, Pini C, Afferni C, Barletta B, Tinghino R, Schininà E, Federico R, Mari A, Di Felice G
Department of Immunology, Istituto Superiore di Sanità, V. la Regina Elena, 299, I-00161 Rome, Italy.
Clin Exp Allergy. 2001 Mar;31(3):458-65. doi: 10.1046/j.1365-2222.2001.01019.x.
Carbohydrate epitopes are capable of binding human IgE from allergic subjects and these epitopes play a role in the cross-reactivity between allergens from unrelated sources. A monoclonal antibody (5E6), specific for a carbohydrate epitope detectable on components of Cupressus arizonica pollen extract, has been produced and characterized. To study the relationship between the epitopes recognized by the monoclonal antibody and by IgE from allergic subjects. To investigate the presence of such carbohydrate IgE determinant in extracts from 21 pollen species belonging to 16 taxonomically related and unrelated families, by means of the monoclonal antibody. IgG-depleted fraction from protein G-purified human allergic serum was obtained. The monoclonal antibody and the IgE from the purified fraction were tested on two glycoproteins, polyamine oxidase and ascorbate oxidase, adsorbed on the ELISA plates. The relationship between the monoclonal- and the IgE-recognized epitopes was investigated by ELISA-competition experiments. Analysis of the distribution of this carbohydrate epitope was performed by direct binding of the monoclonal antibody onto the various extracts. The monoclonal antibody and the IgE were able to bind carbohydrate epitopes on the two plant glycoproteins, ascorbate oxidase and polyamine oxidase. Polyamine oxidase shows only one N-glycosilation site whose carbohydrate moiety seems to be composed of a branched chain of seven ordered sugars, i.e. two N-acetyl-D-glucosamine-, three mannose-, one fucose- and one xylose-residues. This structure bears the epitope recognized by mAb 5E6. Human IgE from the IgG-depleted fraction were found capable of inhibiting the monoclonal antibody binding. The allergenic epitope identified was shared by a large number of extracts with different levels of reactivity (OD490 ranging from 0.110 to 2.060). Our data support the finding that a monoclonal antibody specific for a carbohydrate epitope of Cupressus arizonica pollen extract detects an epitope which is also recognized by IgE from allergic subjects. This characterized reagent could be a useful tool for studying distribution of cross-reactive carbohydrate determinants in allergenic pollen extracts and their components.
碳水化合物表位能够结合过敏患者的人IgE，并且这些表位在来自不相关来源的过敏原之间的交叉反应中起作用。已制备并表征了一种单克隆抗体（5E6），该抗体对在亚利桑那柏花粉提取物成分上可检测到的碳水化合物表位具有特异性。研究单克隆抗体识别的表位与过敏患者IgE识别的表位之间的关系。通过单克隆抗体研究来自16个分类学相关和不相关科的21种花粉提取物中这种碳水化合物IgE决定簇的存在情况。获得了蛋白G纯化的人过敏血清的IgG去除组分。在吸附于ELISA板上的两种糖蛋白，即多胺氧化酶和抗坏血酸氧化酶上测试单克隆抗体和纯化组分中的IgE。通过ELISA竞争实验研究单克隆抗体和IgE识别的表位之间的关系。通过将单克隆抗体直接结合到各种提取物上来分析这种碳水化合物表位的分布。单克隆抗体和IgE能够结合两种植物糖蛋白（抗坏血酸氧化酶和多胺氧化酶）上的碳水化合物表位。多胺氧化酶仅显示一个N-糖基化位点，其碳水化合物部分似乎由七个有序糖的支链组成，即两个N-乙酰-D-葡萄糖胺、三个甘露糖、一个岩藻糖和一个木糖残基。这种结构带有被单克隆抗体5E6识别的表位。发现来自IgG去除组分的人IgE能够抑制单克隆抗体的结合。鉴定出的变应原表位被大量具有不同反应水平（OD490范围为0.110至2.060）的提取物所共有。我们的数据支持以下发现：对亚利桑那柏花粉提取物的碳水化合物表位具有特异性的单克隆抗体检测到一个也被过敏患者的IgE识别的表位。这种经过表征的试剂可能是研究变应原花粉提取物及其成分中交叉反应性碳水化合物决定簇分布的有用工具。
