METAL PORPHYRIN CHEMILUMINESCENCE REACTION AND APPLICATION TO IMMUNOASSAY

A new luminol chemiluminescence chemistry system is described which does not require preparation or use of an oxidizer such as hydrogen peroxide. In the reaction a metal porphyrin catalyzes luminol chemiluminescence in a high pH solution. A comparison of porphyrin catalysts showed that a metal atom is needed in the porphyrin, manganese works better than iron, and substitution of strongly electron withdrawing groups at the para positions of the tetrapyrrole porphine with no substitution of the pyrrole rings gives the best activity. The metal porphyrin chemiluminescence system is enhanced by the presence of unsaturated long-chain fatty acid in the reaction solution. Sensitivity of detection of Mn meso-tetrakis(4-sulfonatophenyl)porphine was 120 amol. Coupling reactions of a carboxyphenyl derivative to antibodies were optimized, and a detection limit of 68 amol was obtained for a metal porphyrin antibody conjugate. An alpha fetoprotein immunoassay developed using the chemiluminescence reaction had a detection limit of 20 pg (0.2 fmol). Good correlation was found (R = 0.99) between immunoassay results and alpha-fetoprotein in 32 human plasma samples. Because the reaction solution needed for methyl porphyrin chemiluminescence is very simple (luminol in NaOH/water) and has good stability, this chemiluminescence detection system may have application to automated assays.