GENERATION OF ANTIBODY-MIMETIC PEPTIDES (MINI-ANTIBODIES) FOR DEVELOPING HIGHLY SENSITIVE AND SPECIFIC BIOANALYTICAL SYSTEMS

抗体作为关键试剂广泛应用于各种生物分析系统中。然而，在动物中引发的常规抗体不仅由于它们的大分子量（约1000）而具有几个缺点。150000），但有时也缺乏足够的亲和力或特异性。因此，具有较小分子量的抗体模拟肽（微型抗体），其将通过基因操作制备，有望成为开发高灵敏度和特异性的生物分析系统的新颖和有用的工具。从这些观点出发，我们制备了两种单链Fv片段（scFv; MW ca. 25000）分别与11-脱氧皮质醇（11-DC，皮质醇的生物合成前体）或（1 R）-丁呋洛尔（β-阻断剂）作用，通过柔性连接肽连接相应小鼠抗体的V_H和V_L结构域。这些DNA片段通过引入接头序列的重叠延伸PCR进行剪接，然后将其亚克隆到表达质粒载体中。用重组体转化大肠杆菌细胞，并从细胞制备作为周质提取物的表达的scFv。RIA或ELISA分析表明，这些scFv具有良好的亲和力和特异性的相关抗原，这是非常相似的原始小鼠抗体，其中V_H-和V_L-序列已衍生。基于这些单链抗体互补决定区的氨基酸序列，有望产生分子量更小的新型分子识别模块。

Antibodies are widely used as a key reagent in various bioanalytical systems. However, conventional antibodies elicited in animals not only possess several disadvantages due to their large molecular weight (ca. 150000), but also sometimes lack enough affinity nor specificity. Thus, antibody-mimetic peptides (mini-antibodies) with a smaller molecular weight, which would be prepared by gene manipulation, are expected to be a novel and useful tools for developing highly sensitive and specific bioanalytical systems. From these points of view, we prepared two kinds of single-chain Fv fragments (scFvs ; MW ca. 25000) against 11-deoxycortisol (11-DC ; the biosynthetic precursor of cortisol) or (1R)-bufuralol (aβ-blocking agent), linking V_H and V_L domains of the corresponding mouse antibody via a flexible linker peptide.The V_H-and V_L-genes were cloned by RT-PCR using the total RNA extracted from the relevant antibody-secreting hybridoma. These DNA fragments were spliced by overlap extension PCR introducing the linker sequence, which was then subcloned into an expression plasmid vector. E.coli cells were transformed with the recombinant, and the expressed scFv was prepared from the cells as a periplasmic extract. An RIA or ELISA analysis revealed that these scFvs possess excellent affinity and specificity to the relevant antigen, which are very similar to those of the original mouse antibodies, from which the V_H-and V_L-sequence has been derived. It is expected that a novel molecular-recognizing module with even smaller molecular weight would be generated based on the amino acid sequence of the complementarity-determining regions of these scFvs.