ИСТИНА

The most sensitive enzyme immunoassay (EIA) is the assay with chemiluminescent (CL) detection of enzyme activity. Traditionally in CL-EIA a light is formed upon the oxidation of luminol with hydrogen peroxide catalyzed by peroxidase using as a label of immunoreagents. Since plant peroxidases are poor catalysts in this reaction, certain compounds known as enhancers are added to the substrate mixture to increase CL intensity. For long time the most popular enhancer for peroxidase was 4-iodophenol. In this work we analyzed some phenothiazine derivatives and showed that phenothiazines carrying groups with negative charge are potent primary enhancers in peroxidase-catalyzed CL, whereas phenothiazines with groups with positive charge have no the enhancing ability. The most efficient primary enhancers, whose the enhancing activity is higher in many times than that of p-iodophenol, are 3-(10’-phenothiazi-nyl)propane-1-sulfonate (SPTZ) [1] and 3-(10’-phenothiazinyl)propionic acid (PPA) [1,2]. As it is shown previously [3], some pyridine derivatives are secondary enhancers increasing the enhancing ability of phenothiazines. Screening of some pyridines showed that N-morpholi-nopyridine (MORPH) is the most active secondary enhancer. The mechanism of its enhancing action was proposed [4]. The conditions of the performance of the enhanced CL reaction using SPTZ or PPA in combination with MORPH as primary and secondary enhancers were optimized. Under these conditions the light intensity was practically unchanged for a long time. The detection systems with SPTZ/PPA and MORPH were applied successfully in construction of ultrasensitive EIA kit for determination of human thyroglobulin [5] and methylglyoxal-modified low density lipoprotein [2]. The obtained results open good perspectives for use of ECR with phenothiazines/MORPH in the development of ultra-sensitive immunoassay kits. The authors thank Dr. Leopoldo Della Ciana (Cyanagen, Italy) for help in the performance of the work and the Russian Foundation for Basic Research for financial support (11-04-92005_NNS_a).