GENERATION OF NOVEL ANTIBODY SPECIES―ANTI-METATYPE ANTIBODIES AND "HYPER-ANTIBODIES" ―AND THEIR APPLICATION TO BIOANALYTICAL SYSTEMS

抗体作为一种关键试剂广泛应用于各种免疫测定或免疫亲和纯化系统中，在生物医学分析中具有重要作用。然而，传统抗体在亲和力和特异性上存在一定的局限性，导致这些免疫化学技术的灵敏度不足或选择性低。在本研究中，我们试图产生新的抗体种类，抗元型抗体（识别抗原-抗体复合物的抗体）和“超抗体”（具有提高亲和力和特异性的基因工程抗体）。这可能使我们能够开发出优秀的生物活性小分子（半抗原）分析系统。特异性识别目标半抗原和抗半抗原抗体之间复合物的抗元型抗体有望实现类固醇和合成药物的非竞争性免疫分析系统，显示出比传统竞争性免疫分析高得多的灵敏度。首先，我们制备了针对石胆酸3-硫酸酯和脱氧胆酸（DCA）的单链Fv片段（scFvs），因为scFvs缺乏免疫球蛋白的恒定区，有利于诱导这类抗元型抗体。利用从杂交瘤细胞中提取的RNA，通过RT-PCR扩增小鼠抗这些胆汁酸单克隆抗体的V_H-和v_l -基因，并将其组装成编码5‘-V_H-(Gly_4-Ser)_3-V_L-3’的scFv序列，在大肠杆菌XLOLR菌株中表达。所得的scFv蛋白对相关半抗原表现出与亲本小鼠抗体相似的反应性。然后，我们研究了与相关半抗原DCA的亲和标记，因为这将有效地抑制半抗原与抗体之间的解离，增加产生抗元型抗体的机会。我们发现dca -腺苷酸对蛋白质上的初级氨基具有高度反应性，并且作为标记剂非常有效。用亲和标记的DCA-anti-DCA scFv复合物免疫小鼠将为我们提供抗元型抗体，从而实现敏感的非竞争性免疫分析程序。少

Antibodies are widely used as a key reagent in various immunoassays or immunoaffinity purification systems, which are essential in the biomedical analyses. However, conventional antibodies have some limitations on both affinity and specificity, resulting in insufficient sensitivity or low selectivity of these immunochemical techniques, In this study, we attempted to generate novel antibody species, anti-metatype antibodies (the antibodies recognizing antigen-antibody complex) and "hyper-antibodies" (the genetically engineered antibodies having improved affinity and specificity), which may allow us to develop excellent analytical systems for bioactive small molecules (haptens).Anti-metatype antibodies that specifically recognize the complex between a target hapten and an anti-hapten antibody are expected to enable noncompetitive immunoassay systems for steroids and synthetic drugs, showing much higher sensitivity than conventional competitive immunoassays. First, we prepared single-chai … More n Fv fragments (scFvs) against lithocholic acid 3-sulfate and deoxycholic acid (DCA) because scFvs lack the constant regions of immunoglobulins and thus would be advantageous to induce such anti-metatype antibodies. The V_H- and V_L-genes of mouse monoclonal antibodies against these bile acids were amplified by RT-PCR using RNA extracted from the corresponding hybridoma cells, which were then assembled into the scFv sequence encoding 5'-V_H-(Gly_4-Ser)_3-V_L-3' and expressed in E. coli XLOLR strain. The resulting scFv proteins showed similar reactivity against the relevant haptens to those of the parent mouse antibodies.Then we investigated the affinity-labeling of the auti-DCA antibody paratope with a relevant hapten, DCA, because this would be effective to inhibit the dissociation between the haptens and antibodies and increase the chance for generating anti-metatype antibodies. We found DCA-adenylate is highly reactive to primary amino groups on proteins, and works very well as the labeling agent. Immunization of mice with the affinity-labeled DCA-anti-DCA scFv complex would provide us the anti-metatype antibodies that enable sensitive noncompetitive immunoassay procedures. Less