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

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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.