ИСТИНА

The most sensitive format of enzyme immunoassay (EIA) is the assay with chemiluminescent (CL) detection of enzyme activity of immunoreagents. Traditionally in CL-EIA horseradish peroxidase (HRP) and 4-iodophenol (PIP) are used as enzyme label and enhancer, respectively. This enhanced chemiluminescence reaction (ECR) based on luminol oxidation has been successfully used in development of ultra-sensitive immunochemical kits for the determination of various compounds. Main drawbacks of the method of peroxidase activity measurement using ECR with HRP/PIP are the relatively quick decay of CL signal and its insufficiently high sensitivity. A replacement of PIP with 3-(10’-phenothiazinyl)propane-1-sulfonate (SPTZ) as the enhancer allowed to increase the intensity of chemiluminescent signal, the light intensity being practically unchanged for a long time. Moreover, a combination of SPTZ and 4-morpholinopyridine (MORPH) allowed additionally the increase of CL no affecting the kinetics of CL decay. The optimization of the experimental conditions for ECR catalyzed by soybean (SbP) and horseradish peroxidases was carried out by a 25 full factorial design. In the case of use of SPTZ/MORPH system a detection limit of SbP was 0.03 pM that was 40-fold lower than that of HRP/PIP system. The SbP/SPTZ/MORPH detection system was applied successfully in construction of ultrasensitive EIA kit for determination of thyroglobulin in human serum. The study showed that a lower detection limit (LOD) of the CL-EIA with SbP/SPTZ/MORPH was 10 times lower than in the assay with HRP/PIP. The obtained results open good perspectives for use of ECR with SPTZ/MORPH in the development of ultra-sensitive immunoassay kits.