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

我们开发了一种新的超灵敏的免疫测定“异捕获夹心EIA”，在amol水平检测等于或小于分子量1KD的半抗原，包括激素干扰物质，能够高灵敏度检测低分子物质。因此，采用噬菌体展示法，对一抗与低分子抗原的复合物制备第二抗体，并利用这两种抗体建立超灵敏非竞争方法。通过检测对蛋白质降解机制的影响，分析激素干扰物质对人体的影响。作为模型半抗原，使用E-64c。该E-64c在体内用巯基蛋白酶抑制剂强烈抑制溶酶体组织蛋白酶B。因此，利用E-64c很好地阐明了胞内降解机制，但其代谢机制尚不明确。在“异捕获夹心EIA”中，我们将E-64c与一抗反应，形成E-64c抗体复合物。使用e -64c抗体结合位点的特异性第二抗体连续测量该复合物。因此采用“噬菌体展示法”制备特异性二抗，但一抗必须为单克隆抗体。然后我们首先决定制备E-64c的单克隆抗体。用碳二亚胺制备E-64c-KLH，免疫小鼠，制备单克隆抗体。目前，我们正在利用噬菌体展示法制备该单克隆抗体/ E-64c结合位点特异性的人工抗体。当我们同时使用两种标记抗体时，我们研究了各种标记化合物和酶标记来逃避位阻。结果表明，从代谢周转率和分子量来看，重组碱性磷酸酶是最合适的，偶联研究认为，马来酰亚胺-铰链法是最合适的。

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.