Development and practical application of enzyme immunoassay of calpastatin

本研究旨在建立钙依赖性半胱氨酸蛋白酶的内源性抑制物--钙调蛋白的酶免疫分析(EL ISA)体系，并评价其临床应用价值。主要研究结果如下：1.抗钙调蛋白抗体的制备--以从猪心和人红细胞中分离纯化的钙调蛋白为免疫原，获得了多克隆抗体和单抗。抗体的特性--我们鉴定了这些抗体在钙调蛋白结构域1和3的不同位置的表位。Calastatin的免疫组织化学--通过免疫组织化学技术，我们在显微镜下显示了calastatin在不同组织中的定位。应用两种不同表位的单抗建立了夹心式酶联免疫吸附试验系统。酶联免疫吸附试验的特性-我们的新的酶联免疫吸附试验使我们能够检测低至100pmol/L的钙调蛋白，并能够区分红细胞型和器官型钙调蛋白。酶联免疫吸附试验的临床应用--我们对血液、体液和组织标本中的钙调蛋白进行了定量，这些样本来自不同疾病的患者，如β-地中海贫血、高血压和关节炎。这种新的酶联免疫吸附试验在临床诊断中具有潜在的实用价值。

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.