Dotsikas Yannis, Loukas Yannis L
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Greece.
Anal Sci. 2012;28(8):753-7. doi: 10.2116/analsci.28.753.
To overcome the sensitivity limit for small molecules (haptens) in immunoassays based on antigen-horseradish peroxidase (HRP) conjugates as labels, a novel approach was established that afforded very low detection limits. Biotinylated anti-HRP antibody was utilized in order to attach, via a streptavidin bridge, liposomaly entrapped HRP. Fentanyl, used as a model antigen, could be determined via the generation of a high-intensity and relatively stable chemiluminescence (CL) signal of a HRP-catalyzed luminol/H(2)O(2)/enhancer system, immediately after the addition of a substrate solution. 4-(1-Imidazolyl)phenol (4-IMP) was used as an enhancer, and the outcome of this combination was a very low detection limit (0.895 pg mL(-1)) in plasma samples. The respective detection limit with the use of just the classical HRP-antigen conjugate was > 5-times higher. Intra- and inter-assay RSDs of the novel assay were 6.8 - 9.9 and 11 - 17%, respectively. The proposed method could be utilized for a wide range of molecules without replacing existing antigen-HRP based kits.
为了克服基于抗原 - 辣根过氧化物酶（HRP）缀合物作为标记物的免疫分析中对小分子（半抗原）的灵敏度限制，建立了一种新方法，该方法具有极低的检测限。利用生物素化抗HRP抗体，通过链霉亲和素桥连接脂质体包裹的HRP。以芬太尼作为模型抗原，在加入底物溶液后，可通过HRP催化的鲁米诺/H₂O₂/增强剂系统产生高强度且相对稳定的化学发光（CL）信号来测定。使用4 -（1 - 咪唑基）苯酚（4 - IMP）作为增强剂，这种组合在血浆样品中的检测限非常低（0.895 pg mL⁻¹）。仅使用经典的HRP - 抗原缀合物时，相应的检测限要高出5倍以上。新方法的批内和批间相对标准偏差分别为6.8 - 9.9%和11 - 17%。所提出的方法可用于多种分子，而无需更换现有的基于抗原 - HRP的试剂盒。
