An amplification system for enzyme immunoassays, in which complexes of streptavidin, biotinylated horseradish peroxidase, and biotinylated anti-horseradish peroxidase antibody are used to detect biotinylated target molecules was developed. In enzyme-linked immunosorbent assay (ELISA) experiments this enzyme complex enhancement (ECA) system gave up to a better than two orders of magnitude increase in sensitivity, compared to a horseradish peroxidase-conjugated streptavidin detection system. The ECA system was shown to he applicable to direct detection of biotinylated antigens, as well as in ELISA employing biotinylated antibodies and in sandwich ELISA. The ECA system was also applicable to immunoblots, giving a sensitivity comparable to that obtained using a radio-chemical detection system. © 1993 Academic Press, Inc.