Enzyme-linked immunosorbent assays based on peroxidase labels and enzyme-amplified lanthanide luminescence detection

The enzyme-amplified lanthanide luminescence (EALL) detection is developed and applied for the determination of peroxidase as marker in enzyme-linked immunosorbent assays (ELISA). The detection scheme is based on the peroxidase catalysed dimerization of 4-hydroxyphenylpropionic acid (pHPPA) and the subsequent formation of a ternary complex with Tb(III)EDTA. Quantum yields and fluorescence lifetimes of the luminescent species are presented to give an estimate of the potential of this procedure. Two different ELISA were performed with the EALL detection scheme. For the first, a model ELISA for the determination of goat anti-rabbit IgG, a limit of determination of 3 micrograms dm-3 (2 fmol) of the antibody could be achieved. As second model assay, a commercial ELISA kit was successfully validated for the new detection scheme. Photometric and EALL detection were in good agreement for the determination of human anti-gliadin IgA in serum.