Analytical Sciences

Improved Performance of Antigen-HRP Conjugate-based Immunoassays after the Addition of Anti-HRP Antibody and Application of a Liposomal Chemiluminescence Marker

Yannis DOTSIKAS and Yannis L. LOUKAS

Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Panepistimioupoli Zografou GR-157 71, Athens, Greece

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/H₂O₂/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.

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