Chemiluminescent measurement of peroxidase activity and its application using a lucigenin CT-complex.
Luminol (3-aminophthaloylhydrazine) is well known as a chemiluminescent reagent that can be used to measure peroxidase (POD) activity. We have developed a more sensitive assay for POD using a new chemiluminescent reagent based on 10,10'-dimethyl-9, 9'-biacridinium nitrate (lucigenin). This new reagent was obtained from lucigenin by light irradiation in an organic polar solvent such as N,N-dimethylacetamide (DMAc). The detection limit for POD activity using this reagent is 10(-19) mol/assay. In an application for enzyme immunoassay based on a POD label, we were able to detect human alpha-fetoprotein (AFP) at 1pg/mL. The characterization of this chemiluminescent reagent was obtained by optical measurements, and the formation of lucigenin charge transfer complex (CT-complex) was confirmed. We also investigated the stability of lucigenin CT-complex as a chemiluminescent reagent for the measurement of POD activity, and confirmed that the reagent was stable for more than a year.
Katsuragi
H
H
Technical Research Center, Dainichi-Seika Color and Chemicals Manufacturing Co. Ltd., 1-9-4 Horinouchi, Adachi-ku, Tokyo 123-8555, Japan.
Takahashi
K
K
Suzuki
H
H
Maeda
M
M
D000166:Acridines; D000911:Antibodies, Monoclonal; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D007124:Immunoenzyme Techniques; D008163:Luminescent Measurements; D008165:Luminol; D010544:Peroxidases / Q000032:analysis*; D012680:Sensitivity and Specificity; D000509:alpha-Fetoproteins / Q000032:analysis*
D000166:Acridines; D000911:Antibodies, Monoclonal; D000509:alpha-Fetoproteins; C033472:10,10'-dimethyl-9,9'-biacridinium; D008165:Luminol; D010544:Peroxidases