Generation of anti-idiotype antibodies as the novel reagents for highly sensitive determination of small biomolecules

对小分子(半抗原)，如类固醇和合成药物的常规免疫分析依赖于未标记的抗原(分析物)和标记的抗原与有限数量的抗体之间的竞争反应。尽管非竞争性免疫分析方法，如两点免疫分析，提供了更高的灵敏度(阿托摩尔-齐托摩尔顺序)和更宽的工作范围，但这一原理并不适用于半抗原的测量。因此，我们试图建立一种以抗独特型抗体为关键试剂，以熊去氧胆酸7-N-乙酰氨基葡萄糖(UDCA 7-NAG；一种胆汁酸代谢物)和I 1-脱氧皮质醇(11-DC)为模型半抗原的非竞争性免疫分析系统。用与锁孔花青素偶联的半抗原免疫BALB/c或A/J小鼠。免疫脾细胞与P3INS L/L-Ag4-1骨髓瘤细胞融合，可获得A型和β型抗独特型单抗，分别识别抗半抗原抗体的骨架区域和副表位。选择的α-型和β-型抗体与抗半抗原抗体相结合，提供了一种灵敏的UDCA7-NAG非竞争性检测系统，其测量范围约为0.2-500pg，最小检出量为130fg(=220amol)。

Conventional immunoassays for small molecules (haptens) such as steroids and synthetic drugs are dependent on the competitive reaction between an unlabeled antigen (analyte) and labeled-antigen against a limited amount of antibody. Although noncompetitive immunoassay procedures such as two-site immunometric assays offer a much higher sensitivity (attomole-zeptomole order) and a wider working range, this principle has not been suitable for measuring haptens. Thus, we attempted to develop a noncompetitive immunoassay system using anti-idiotype antibodies as the key reagent ; ursodeoxycholic acid7-N-acetylglucosaminide (UDCA 7-NAG ; a bile acid metabolite) and I 1-deoxycortisol (11-DC) were selected as the model hapten. BALB/c or A/J mice were immunized with amonoclonal antibody against the hapten which had been conjugated with keyhole limpethemocyanin. The fusion between the immune spleen cells and P3INS l/l-Ag4-1 myeloma cells afforded a-type and beta-type monoclonal anti-idiotype antibodies, each recognizing the framework region and paratope of the anti-hapten antibody. The combined use of selected alpha-type and beta-type antibodies together with the anti-hapten antibody provided a sensitive noncompetitive assay system of UDCA 7-NAG, whose measurable range was approximately0.2- 500 pg, and the minimum detectable amount was 130 fg (= 220 amol).