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

抗体被广泛用作各种免疫分析或免疫亲和纯化系统中的关键试剂，这在生物医学分析中是必不可少的。然而，传统的抗体在亲和力和特异性上都有一定的局限性，导致这些免疫化学技术的灵敏度不够或选择性低。在本研究中，我们试图产生新的抗体种类、抗变型抗体(识别抗原-抗体复合体的抗体)和“超抗体”(具有更高亲和力和特异度的基因工程抗体)，这可能使我们开发出优秀的生物活性小分子(半抗原)分析系统。首先，我们准备了单茶…更多的抗石胆酸、3-硫酸盐和脱氧胆酸(DCA)的单链抗体(ScFv)，因为scFv缺乏免疫球蛋白的恒定区，因此有利于诱导这种抗元型抗体。从相应的杂交瘤细胞中提取RNA，通过RT-PCR扩增出小鼠抗胆汁酸单抗的V_H-和V_L-基因，将其组装成编码5‘-V_H-(Gly_4-Ser)_3-V_L-3’的单链抗体序列，并在大肠杆菌XLOLR中表达。然后，我们研究了Auti-DCA抗体副表位与相关半抗原DCA的亲和标记，因为这将有效地抑制半抗原和抗体之间的解离，并增加产生抗Metatype抗体的机会。我们发现DCA-腺苷对蛋白质上的伯氨基具有很高的反应性，是一种很好的标记剂。用亲和标记的DCA-抗-DCA单链抗体免疫小鼠，将为我们提供抗Metatype抗体，使敏感的非竞争免疫分析程序成为可能。较少

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