Pulli Timo, Höyhtyä Matti, Söderlund Hans, Takkinen Kristiina
VTT Biotechnology, Tietotie 2, P.O. Box 1500, FI-02044 VTT, Espoo, Finland.
Anal Chem. 2005 Apr 15;77(8):2637-42. doi: 10.1021/ac048379l.
We have developed a one-step, homogeneous noncompetitive immunoassay for small analytes using recombinant antibodies and morphine as the model analyte. A highly specific antibody against the immune complex (IC) formed between an anti-morphine antibody and morphine was selected from a naive scFv phage display library. The in vitro phage library selection procedure avoids the difficulties associated with the production of anti-IC antibodies by animal immunization. The anti-morphine and the anti-IC antibodies were labeled with a pair of fluorescence resonance energy transfer (FRET) fluorophores. In the FRET assay the labeled antibodies were incubated with saliva samples spiked with morphine, codeine, or heroin. Within 2 min, 5 ng/mL morphine, which is clearly under the recommended cutoff level, was detected without cross-reactivity to codeine or heroin. This assay principle is also widely applicable to other small analytes.
我们开发了一种使用重组抗体和吗啡作为模型分析物的针对小分子分析物的一步法均相非竞争性免疫测定法。从天然单链抗体噬菌体展示文库中筛选出一种针对抗吗啡抗体与吗啡形成的免疫复合物（IC）的高特异性抗体。体外噬菌体文库筛选程序避免了通过动物免疫产生抗IC抗体所带来的困难。抗吗啡抗体和抗IC抗体用一对荧光共振能量转移（FRET）荧光团进行标记。在FRET测定中，将标记的抗体与添加了吗啡、可待因或海洛因的唾液样本一起孵育。在2分钟内，检测到浓度为5 ng/mL的吗啡，该浓度明显低于推荐的临界值水平，且对可待因或海洛因无交叉反应。该测定原理也广泛适用于其他小分子分析物。
