Employment of 4-(1-imidazolyl)phenol as a luminol signal enhancer in a competitive-type chemiluminescence immunoassay and its comparison with the conventional antigen–horseradish peroxidase conjugate-based assay

This study describes the employment of a novel imidazole-substituted phenol [4-(1-imidazolyl)phenol] as a highly potent signal enhancer in a horseradish peroxidase (HRP)–luminol chemiluminescence (CL) immunoassay. This competitive-type immunoassay for the model antigen fentanyl is based on the use of fentanyl polyclonal antibody immobilized on white microtiter plates and a biotinylated bovine serum albumin (BSA)–fentanyl derivative as a tracer. The latter was detected by means of streptavidin labeled with HRP, resulting in the generation of a high-intensity and relatively stable chemiluminescent signal, immediately after the addition of the substrate solution (NOAS). The developed method fulfilled the requirements of accuracy (percentage recovery ranged from 93.8 to 107%) and precision (intra- and interassay CVs were 2.5–5.2 and 4.5–11.9%, respectively). Its plasma detection limit (1.05 pg ml^-1) was lower than those of previous immunoassays. The novel assay was compared in terms of sensitivity and concentration range with other common HRP substrate systems: luminol–p-iodophenol–H2O2 and TMB–H2O2. Finally, the described method was compared with an HRP–fentanyl conjugate-based assay, similar to commercially available kits (SKIT), employing the novel substrate solution for both assays and the differences observed were explained by applying previously described models. The detection limit was 4.82 pg ml^-1 for SKIT, recovery values were 94.2–105% and intra- and inter-assay CVs were 2.5–5.2 and 4.5–11.9%, respectively. In conclusion, the proposed assay could be utilized for a wide range of molecules and replace the existing enzyme-labeled antigen-based kits.