Optimization of horseradish peroxidase-catalyzed enhanced chemiluminescence reaction by full factorial design

Using a full factorial design the optimization of experimental conditions of enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP-C) in the presence of 3-(10′-phenothiazinyl)propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORP) as enhancers was performed. The effect of concentrations of SPTZ, hydrogen peroxide, MORP, luminol, and Tris on a ratio of peroxidase-catalyzed CL to background was studied. The use of the full 25 factorial design instead of “one-variable-a time” method allowed to increase the sensitivity of HRP-C determination 2355 fold without a change of detection limit. The obtained results open up very promising perspectives for using HRP-C-catalyzed ECR to improve the sensitivity of chemiluminescent enzyme immunoassay.