Improved performance of antigen-HRP conjugate-based immunoassays after the addition of anti-HRP antibody and application of a liposomal chemiluminescence marker.

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.