Development of "heterocapture sandwich EIA" and detection of the a hormone-disrupting substance

我们开发了新的超灵敏免疫测定法“异源捕获夹心EIA”，可检测分子量等于或小于1 KD的半抗原抗原，并在其水平上，包括破坏酶活性的物质，从而能够高灵敏度检测低分子物质。因此，利用噬菌体展示方法，制备第一抗体与低分子抗原的复合物的第二抗体，并通过这两种抗体建立超灵敏的非竞争性方法。为了分析蛋白质降解机制的影响，我们使用E-64 c作为模型半抗原，研究了蛋白质降解机制的影响。该E-64 c在体内与巯基蛋白酶抑制剂一起强烈抑制溶酶体中的组织蛋白酶B。因此，E-64 c可以很好地用于阐明细胞内降解机制，但代谢机制不明显。在“异源捕获夹心EIA”中，我们使E-64 c和一抗反应并形成E-64 c-抗体复合物。使用E-64 c-抗体结合位点的特异性第二抗体连续测量该复合物。因此，利用噬菌体展示技术制备特异性的第二抗体，但第一抗体必须是单克隆抗体。因此，我们首先决定制备针对E-64 c的单克隆抗体。用碳二亚胺法制备E-64 c-KLH，免疫小鼠制备单克隆抗体。目前，我们正在通过噬菌体展示方法制备对该单克隆抗体/ E-64 c结合位点具有特异性的人工抗体，同时使用两种标记抗体时，我们考察了各种标记化合物和酶标记以避免空间位阻。结果，从代谢周转率和分子量来看，重组碱性磷酸酶是合适的，并且接合理解为马来酰亚胺铰链方法是最合适的。

We develop new ultra sensitive immunoassay "heterocapture sandwich EIA" detecting a haptenic antigen equal to or less than molecular weight 1KD at an amol level and, including a hormone-disrupting substance, enable high sensitive detection of the low-molecular substance. Therefore, using a phage display method, it prepares second antibody for the complex of the primary antibody with a low molecule antigen and establishes ultra sensitive non-competitive method by these two kinds of antibodies. We examined the influence on protein degradation mechanism to analyze the influence on human body by the a hormone-disrupting substance.As a model hapten, it was used E-64c. This E-64c strongly inhibits cathepsin B in lysosome with thiol protease inhibitor in vivo. Therefore, E-64c is used for the elucidation of the intracellular degradation mechanism well, but the metabolism mechanism is not apparent. In "the heterocapture sandwich EIA", we react E-64c and primary antibody and form an E-64c-antibody complex. This complex is measured using specific second antibody for the E-64c-antibody combining site continuously. So it prepares specific second antibody by "a phage display method", but primary antibody must be monoclonal antibody. Then at first we decided to prepare monoclonal antibody for E-64c. We made E-64c-KLH using carbodiimide and immunized it to mouse and prepared monoclonal antibody. Now, we are making the artificial antibody which is specific for this monoclonal antibody / E-64c binding site by a phage display method.We examined various kinds of labeled compounds and enzymatic tagging to evade steric hindrance when we used two kinds of labeled antibody in parallel with this. As a result, recombinant alkaline phosphatase was suitable from a metabolic turnover rate and molecular weight, and conjugation understood that a maleimide-hinge method was suitable most.