Development and practical application of enzyme immunoassay of calpastatin

本研究旨在建立一种钙蛋白酶抑制蛋白的酶免疫分析方法。例如，钙依赖性半胱氨酸蛋白酶（钙蛋白酶）的内源性抑制剂，及其临床价值的评价。主要研究结果如下：1.抗钙蛋白酶抑制剂抗体的制备以猪心和人红细胞中的钙蛋白酶抑制剂为免疫原，分别获得了多克隆抗体和单克隆抗体.抗体的表征--我们在钙蛋白酶抑制蛋白结构域1和3的不同位点鉴定了这些抗体的表位。钙蛋白酶抑制蛋白免疫组化--应用免疫组化技术，我们在显微镜下显示了钙蛋白酶抑制蛋白在各种组织中的定位.钙蛋白酶抑制蛋白酶联免疫吸附试验（ELISA）的建立--采用两种具有离散表位的单克隆抗体建立了一种双抗体夹心ELISA体系. ELISA的特性--我们的新ELISA使我们能够测量低至<100 pmol/L的calpastatin，并区分红细胞型和器官型calpastatin。ELISA的临床应用--我们定量检测了各种疾病患者的血液、体液和组织标本中的钙蛋白酶抑制蛋白，如β-地中海贫血、高血压和关节炎。钙蛋白酶水平的疾病特异性变化表明这种新的ELISA用于临床诊断的潜在有用性。

This study aimed at the development of an enzyme immunoassay (ELISA) system for calpastatin, i. e., an endogenous inhibitor of a Ca^<2+>-dependent cysteine protease (calpain), and the evaluation of its clinical value. Our main findings are sununarized as follows :1. Preparation of anti-calpastatin antibodies -- By using calpastatins purified from porcine heart and human erythrocytes as immunogens, we obtained polyclonal as well as monoclonal antibodies.2. Characterization of antibodies -- We identified the epitopes of these antibodies at various sites of domain 1 and 3 of calpastatin.3. Immunohistochemistry of calpastatin -- By immunohistochernical techniques, we showed the localization of calpastatin in various tissues at a rm'croscopic, level.4. Development of ELISA of calpastatin -- A sandwich-type ELISA system was developed employing two monocloiial antibodies with discrete epitopes.5. Characterization of ELISA --- Our new ELISA enabled us to measure calpastatin as little as<100 pmol/L and also to differentiate between erythrocyte-type and organ-type calpastatins.6. Clinical application of ELISA -- We quantitated calpastatin in blood, body fluid and tissue specimens obtained from patients of various diseases, such as beta-thalassemia, hypertension, and arthritis. Disease-specific changes in the calpain level suggested potential usefulness of this new ELISA for clinical-diagnosis.