肥大细胞表面抗原-1高灵敏度定量分析法的建立

A highly sensitive method for quantitation of mast cell surface antigen-1

目的建立肥大细胞表面抗原-1(MASA-1)的高灵敏度定量分析方法。方法首先制备抗人MASA-1的单克隆抗体,然后结合先前制备的多克隆抗体,通过优化包被浓度、温度和反应时间等条件,建立夹心酶联免疫定量方法。结果成功制备了特异性高的小鼠抗人MASA-1的单克隆抗体;夹心ELISA法的检测灵敏度可达0.5 ng/ml。利用该法测出各种呼吸系统疾病病人的肺清洗液中的MASA-1含量为0～130 ng/ml。MASA-1的浓度与甲苯胺蓝染色阳性细胞数之间不存在相关关系,但与MASA-1染色阳性细胞数之间呈显著正相关关系。结论相对于MASA-1细胞染色法,本研究开发的酶联免疫法具有快速、准确及能同时分析大量样品的优点,有助于肥大细胞的实验室研究和临床检测。

Mast cell plays a pivotal role in type I allergy, innate immunity, chronic inflammation and tissue remodeling. The recently cloned mast cell surface antigen-1 （MASA-1） protein is mainly expressed on the surface of activated mast cells, and is useful in the determination of the activation status of mast cells. The aim of this study is to develop a rapid and sensitive method for the quantitation of MASA-1. Based previously prepared a mouse monoelonal antibody against human MASA-1, we optimized the conditions including coating concentration, temperature and incubation time, and finally established a sandwich ELISA with a sensitivity of 0.5 ng/ml. By using this method, the MASA-1 levels in the bronchoalveolar lavage fluid of patients with respiratory system disorders are measured to be within the range of 0-130 ng/ml. The MASA-1 level correlates with the cell number of MASA-1- positive cells, but not with that of cells stained by toluidine blue. Compared with the cell staining technique, the ELISA system is more rapid and accurate, and allows handling of large number of same samples in parallel, therefore is applicable in both research study and clinical test relating to mast cell.