STUDIES ON GENERATION OF NOVEL ANTIBODY MOLECULES FOR MOLECULAR RECOGNITION

抗生素被广泛用作各种免疫测定或免疫亲和性净化系统中的关键试剂，它们是生物医学分析中的基本试剂。根据该报告，传统抗体对亲和力和特殊性都有一定的限制，在不充分的敏感性或这些免疫化学技术的选择性较低的结果。在本研究中，我们试图生成新的抗体分子，我们可以使用超敏感性和特殊性开发优秀的分析系统。α型和β型单克隆抗歧型抗体，每个都可以识别出由B细胞杂交瘤技术产生的抗突变抗体的框架区域和参数，为了开发一个高度敏感的非竞争性免疫测定系统，该系统已经变得很难为Haptens建立。Ursodeoxycholic Acid 7-N-乙酰葡萄糖胺（UDCA 7-NAG）和11-deoxycortisol（11-DC）被选择为Hapten模型。α-和β-型抗异性恋抗体结合使用，提供抗结合抗体的高敏感性测定系统：UDCA 7-NAG的检测限值为70 fg （118 amol），11-DC的1 pg （2.9 fmol）。一项交叉反应研究揭示了这两种检测系统都有实际的特性。UDCA 7-NAG非竞争性评估系统已得到良好验证，可用于直接测量（没有任何预先处理）这个代谢物的人类泌尿系统水平。在另一边，单链Fv片段（scFv）对抗11-DC，显示出非常高的亲和力（Ka 1.3 x10 y D110 y D1 M y D1-1 y D1） and specificity （cross-reactivity with cortisol，0.14%， cortisone 0.21%）， has been clones。如果在这种scFv和绿色荧光蛋白之间克隆融合蛋白，将启用一个敏感的荧光免疫测定系统，现在将在我们的实验室中继续生长。

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 attempt to generate novel antibody molecules, which may allow us to develop excellent analytical systems with ultrahigh sensitivity and specificity.α-type and β-type monoclonal anti-idiotype antibodies, each recognizing the framework region and paratope of an anti-hapten antibody have been produced by the B-cell hybridoma technique, for developing a highly sensitive noncompetitive immunoassay system which has been difficult to establish for haptens. Ursodeoxycholic acid 7-N-acetylglucosaminide (UDCA 7-NAG) and 11-deoxycortisol (11-DC) were selected as the model hapten. The combined use of the α- and β-type anti-idiotype antibodies together with the anti-hapten antibody provided a highly sensitive assay system : the detection limit was 70 fg (118 amol) for UDCA 7-NAG and 1 pg (2.9 fmol) for 11-DC. A cross-reaction study revealed that both assay systems have practical specificity. The UDCA 7-NAG noncompetitive assay system has already been well validated to be available for direct measurement (without any pretreatment) of human urine levels of this metabolite.On the other hand, a single-chain Fv fragment (scFv) against 11-DC, which shows very high affinity (Ka 1.3 x 10ィイD110ィエD1 MィイD1-1ィエD1) and specificity (cross-reactivity with cortisol, 0.14%, cortisone 0.21%), has been clones. Cloning of a fusion protein between this scFv and green fluorescent protein, which would enable a sensitive fluoroimmunoassay system, is now ongoing in our laboratory.