Enzyme-labeled Antigen Method: Development and Application of the Novel Approach for Identifying Plasma Cells Locally Producing Disease-specific Antibodies in Inflammatory Lesions.
Enzyme-labeled Antigen Method: Development and Application of the Novel Approach for Identifying Plasma Cells Locally Producing Disease-specific Antibodies in Inflammatory Lesions.
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DOI：
10.1267/ahc.15030
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发表时间：
2016-02-27
影响因子：
2.4
通讯作者：
Tsutsumi Y
中科院分区：
文献类型：
--
作者：
Mizutani Y;Shiogama K;Onouchi T;Sakurai K;Inada K;Tsutsumi Y
在自身免疫性疾病和感染性疾病的慢性炎性病变中，经常观察到浆细胞。由浆细胞产生的抗体所识别的抗原大多尚不清楚。一种识别这些相应抗原的新技术可能会给我们从病理生理学的角度理解疾病带来突破，仅仅因为免疫细胞在病变内可见。我们开发了一种酶标记抗原方法，用于显微镜下识别炎症病变浆细胞中局部产生的特异性抗体识别的抗原。首先，通过小麦无生殖细胞蛋白合成系统或化学生物素化构建目标生物素化抗原。接下来，筛选组织提取物中对抗体有反应的蛋白，并通过alphasgreen方法评估抗体滴度。最后，采用酶标记抗原法，以生物素化抗原为探针，将产生特异性抗体的浆细胞显微镜定位在固定的冷冻切片上。我们的新方法显示了组织浆细胞产生的抗体：1)类风湿性关节炎自身抗体，2)牙周炎或根状囊肿牙龈卟卟单胞菌主要抗原抗体，3)复发性扁桃体炎化脓性链球菌碳水化合物抗原链球菌a抗体。评估局部特异性抗体反应有望有助于澄清炎性疾病中先前未知的过程。
In chronic inflammatory lesions of autoimmune and infectious diseases, plasma cells are frequently observed. Antigens recognized by antibodies produced by the plasma cells mostly remain unclear. A new technique identifying these corresponding antigens may give us a breakthrough for understanding the disease from a pathophysiological viewpoint, simply because the immunocytes are seen within the lesion. We have developed an enzyme-labeled antigen method for microscopic identification of the antigen recognized by specific antibodies locally produced in plasma cells in inflammatory lesions. Firstly, target biotinylated antigens were constructed by the wheat germ cell-free protein synthesis system or through chemical biotinylation. Next, proteins reactive to antibodies in tissue extracts were screened and antibody titers were evaluated by the AlphaScreen method. Finally, with the enzyme-labeled antigen method using the biotinylated antigens as probes, plasma cells producing specific antibodies were microscopically localized in fixed frozen sections. Our novel approach visualized tissue plasma cells that produced 1) autoantibodies in rheumatoid arthritis, 2) antibodies against major antigens of Porphyromonas gingivalis in periodontitis or radicular cyst, and 3) antibodies against a carbohydrate antigen, Strep A, of Streptococcus pyogenes in recurrent tonsillitis. Evaluation of local specific antibody responses expectedly contributes to clarifying previously unknown processes in inflammatory disorders.