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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。
Objective To establish a high-sensitivity quantitative assay for Mast cell surface antigen-1 (MASA-1). Methods Monoclonal antibody against human MASA-1 was firstly prepared, and then the polyclonal antibody prepared previously was used to establish a sandwich enzyme-linked immunosorbent assay (ELISA) by optimizing the conditions of coating concentration, temperature and reaction time. Results The specific monoclonal antibody against human MASA-1 was successfully prepared. The sensitivity of sandwich ELISA was 0.5 ng / ml. Using this method, MASA-1 levels in lung washes of patients with various respiratory diseases were measured at 0-130 ng / ml. There was no correlation between the concentration of MASA-1 and the number of toluidine blue staining positive cells, but there was a significant positive correlation between the number of MASA-1 positive cells and MASA-1 staining. Conclusion Compared with the MASA-1 cell staining method, the ELISA developed in this study has the advantages of rapid, accurate and simultaneous analysis of a large number of samples, which is helpful for the laboratory research and clinical detection of mast cells.