Sensitivity of immunoenzymometric assay and detection method of enzyme

The effect of the detection limit of an enzyme measurement on the detection limit of the immunoenzymometric assay (IEMA) was investigated. Using a biotin-labelled antibody and avidin-biotin alkaline phosphatase complex (ABC enzyme) reagent, three IEMA systems for interferon-gamma with different enzyme substrates for colorimetric, fluorometric, or chemiluminometric detection were developed. The optimum amounts of the reagents, the non-specific binding (NSB) level, and the detection limit of the IEMA were estimated. The results of this study suggest that the biotin-labelled antibody and ABC enzyme reagent should not decrease to less than 20 times the concentration of its Kd value and to less than 250 times the enzyme activity of the NSB, respectively. The detection limit of IEMA did not decrease as much as that of enzyme measurement because of lack of proportionate decrease of the NSB level. These findings should be very useful not only for IEMA but also for immunoblotting and immunocytochemistry research.