title: Improved performance of antigen-HRP conjugate-based immunoassays after the addition of anti-HRP antibody and application of a liposomal chemiluminescence marker.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Greece.
authors.forename: Yannis
authors.initials: Y
authors.lastname: Dotsikas
authors.forename: Yannis L
authors.initials: YL
authors.lastname: Loukas
mesh_terms: D000906:Antibodies / Q000276:immunology*; D000941:Antigens / Q000097:blood / Q000737:chemistry / Q000276:immunology*; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D057230:Limit of Detection; D008081:Liposomes / Q000737:chemistry*; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods*
chemical_list: D000906:Antibodies; D000941:Antigens; D008081:Liposomes; D006735:Horseradish Peroxidase