Highly sensitive immunosensing based on new generation antibodies against a hapten-acceptor complex

生理活性小分子(半抗原)的示踪表征是生物医学分析中的一个重要课题。遗憾的是，由于半抗原分子质量较低，非竞争性双点免疫测定方法不适用于半抗原，导致难以获得亚分子灵敏度。为了克服这一局限，我们设计了一种新的方法，利用与目标半抗原(受体；ACC)具有亲和力的小分子和针对半抗原-ACC复合体的抗体：这种抗体有望实现对半抗原的高灵敏免疫计量型传感系统。β-环糊精(β-CD)被选为ACC，25-羟基维生素D_3、9-顺式维甲酸和酚酞(PP)作为模型半抗原。我们从突变的展示单链抗体的噬菌体文库中分离出显示单链抗体片段(ScFv)的噬菌体克隆，在有半抗原存在的情况下，其结合强度是没有半抗原时的2~4倍。其中一个抗PP克隆的信号强度随着PP添加量的增加而增加(从4到100nmol)，这表明所展示的ScFv可用于预期的半抗原免疫传感系统。然后我们尝试使用单域抗体(sdAb；仅由V_H结构域组成的抗体片段)作为ACC。构建了小鼠抗雌二醇(E_2)抗体突变的V_H结构域文库，并分离到了一些与E_2结合的克隆。我们现在正计划通过引入更多的点突变来创造对E_2具有更高亲和力的改良sdAbs。由于sdAbbs具有比CDs更高的亲和力和特异性，因此sdAbbs有望在免疫传感系统中成为更实用的ACC。

Trace characterization of physiologically active small molecules (haptens) is an important subject in biomedical analysis. Unfortunately, noncompetitive two-site immunometric assays are not applicable to the haptens, because of their low molecular mass, resulting in the difficulty in gaining subfemtomole sensitivity. To overcome this limitation, we planed a new approach employing the small molecules showing an affinity to the target haptens (acceptors; ACC) and engineered antibodies specific to the hapten-ACC complexes : such antibodies are expected to enable highly sensitive immunometric-type sensing systems for haptens.β-Cyclodextrin (β-CD) was selected as the ACC, and 25-hydroxyvitaminD_3, 9-cis-retinoic acid and phenolphthalein (PP) were as model haptens. We isolated the phage clones displaying the single-chain Fv fragments (scFvs) that exhibit ca. 2〜4-fold stronger binding in the presence of hapten than the absence for each hapten from a mutated scFv-displaying phage library. One of these clones selected against PP afforded increasing signal intensity with the increase in the amount of added PP (ranging from 4 to 100 nmol) suggesting the availability of the displayed scFv for the expected hapten immunosensing systems. We then attempted to use the single-domain antibody (sdAb ; the antibody fragments that consist of only V_H domain) as ACC. A library of mutated V_H domains derived from a previously established mouse anti-estradiol (E_2) antibody was constructed, and some clones showing the binding to E_2 immobilized on microplate were isolated. We are now planning to create the improved sdAbs showing higher affinity to E_2 by introducing further point mutations. Because sdAbs would possess higher a affinity and specificity to a target than CDs, sdAbs are expected to be more practical ACC in the immunosensing systems.