Which of the commonly used marker enzymes gives the best results in colorimetric and fluorimetric enzyme immunoassays: horseradish peroxidase, alkaline phosphatase or beta-galactosidase?

Abstract

Comparing the marker enzymes horseradish peroxidase (HRP), alkaline phosphatase (AP) and beta-galactosidase (beta Gal) in IgG-coupled form with respect to their temperature-dependent kinetics over a period of 22 h the temperature of 37 degrees C warrants highest substrate turnover for all enzymes at all reaction times using fluorogens. Also applying chromogens the optimum temperature for beta Gal is 37 degrees C and depends for HRP and AP on the reaction time. The substrate turnover of HRP using ABTS as chromogen is much higher compared to the other enzymes--both related to mol enzyme (molar activity) and to gram enzyme (specific activity). The turnover decreases for all enzymes in different degrees after coupling to IgG. The turnover of fluorogenic substrates is lower for all enzymes than the turnover of chromogenic substrates but due to the more sensitive detection of fluorogenic products the detection limits for all conjugates were lowered too--especially for beta Gal-IgG by a factor of 333 compared to the colorimetric procedure. In a 2-site binding enzyme immunoassay for alpha-1-fetoprotein (AFP) the detection limit for AFP was reduced by a factor of 2 only by the fluorimetry compared to the colorimetry with all 3 marker enzymes. The HRP-IgG conjugates warranted lowest detection limits for AFP (0.5-1 microgram/1), highest analytical sensitivity (slope of standard curves) at shortest periods of substrate reaction compared to the other enzymes.