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免疫分析方法“异种捕获夹心EIA”，可以在氨基水平上检测分子质量等于或小于1KD的半抗原，并且包括一种激素干扰物质，能够实现对低分子物质的高灵敏度检测。因此，采用噬菌体展示的方法，为一抗与低分子抗原的复合体制备二抗，并利用这两种抗体建立了超灵敏的非竞争性方法。为了分析α-激素干扰物质对人体的影响，我们考察了它对蛋白质降解机制的影响，并以E-64c为模型半抗原。该E-64c在体内用硫醇蛋白水解酶抑制剂强烈抑制溶酶体中的组织蛋白酶B。因此，E-64c可以很好地用于阐明细胞内的降解机制，但代谢机制并不明显。在异源捕获夹心EIA中，我们将E-64c与一抗反应，形成E-64c-抗体复合体。用针对E-64c抗体结合位点的特异性二抗连续测定该复合体。因此，用“噬菌体展示法”制备特异性二抗，但一抗必须是单抗。然后，我们首先决定制备抗E-64c的单抗。用碳化二亚胺制备E-64c-KLH，免疫小鼠，制备单抗。现在，我们正在通过噬菌体展示的方法制备针对该单抗/E-64c结合位点的人工抗体，同时使用两种标记抗体时，我们检测了各种标记化合物和酶标记以避免空间位阻。结果表明，从代谢转化率和相对分子质量的角度来看，重组碱性磷酸酶是最合适的方法，而结合分析则认为马来酰亚胺-铰链法最合适。

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.