Development of clinical assay method for human chymase

该项目的最初目的是1）开发血浆人糜酶的Westernblot分析和生化测定，2）确定血浆糜酶测定的临床意义，3）开发人糜酶的RIA方法以处理大量临床样本，4）比较各种心血管疾病中的血浆糜酶水平，5）确定血浆糜酶水平与各种心血管疾病之间的关系。其中，目标1和目标2已在项目第一年内完成，但目标3因碘化重组h-糜酶不与特异性抗体结合而暂停。因此，我们建立了双夹心ELISA使用单克隆抗体和多克隆抗体的重组人糜酶。血浆糜酶水平在全身性炎症性疾病（肺炎、自身免疫性疾病等）中显著升高。在动脉粥样硬化和缺血性心脏病患者中观察到轻度增加。血浆糜酶水平与血浆c反应蛋白、纤维蛋白原呈显著正相关。提示血浆糜酶水平可反映组织炎症程度。这些发现已在实验生物学会议（华盛顿DC，美国）上报告，并正在考虑在同行评审期刊上发表。为了将这种ELISA应用于临床，需要提高灵敏度。

The original objectives of the project was 1) to develop Westernblot analysis and biochemical assay for plasma human chymase, 2) to determine clinical significance of plasma chymase measurement, 3) to develop RIA method for human chymase to handle massive clinical samples, 4) to compare plasma chymase levels in various cardiovascular diseases, 5) to determine the association between plasma chymase level and various cardiovascular diseases. Among these objectives, objectives 1 and 2 were completed within the first project year, but objectives 3 has been suspended because the iodinated recombinant h-chymase did not bind with the specific antibody. Therefore, we establish a double sandwich ELISA using monoclonal and polyclonal antibodies for recombinant human chymase. Plasma chymase levels significantly increased in systemic inflammatory diseases (pneumonia autoimmune disease and etc.) Mild level of increase was observed in patients with atherosclerosis and ischemic heart disease. There was a significant positive correlation between plasma chymase level and plasma c-reactive protein or fibrinogen. This fact suggests that plasma chymase level may reflect the levels of tissue inflammation. These findings have been reported in Experimental Biology Meeting (Washington DC, USA) and are under considerations for publication in peer review journal. In order to apply this ELISA to clinical use, improvement of the sensitivity will be necessary.