Development of ultra-sensitive soybean peroxidase-based CL-ELISA for the determination of human thyroglobulin

Serum thyroglobulin (Tg) is a main marker of thyroid cancer relapses after total or near-total thyroidectomy of patients with differentiated thyroid carcinoma. In this study, we developed a chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for detecting Tg in human serum. Soybean peroxidase (SbP) in combination with 3-(10'-phenothiazinyl)propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORPH) and horseradish peroxidase (HRP) with p-iodophenol (PIP) were used as detection systems in the sandwich CL-ELISA. Comparison of these two systems showed that a lower detection limit (LOD) of CL-ELISA with SbP/SPTZ/MORPH was 10 times lower than for the immunoassay with HRP/PIP. The LOD value for SbP-based CL-ELISA of 0.2 ng/mL was identical to LOD value typical of CL-ELISA Immulite kit produced with alkaline phosphatase. The sensitivity of Tg CL-ELISA using SbP/SPTZ/MORPH completely satisfies the requirements of modern endocrinology. Comparative study of clinical serum specimens assayed by the SbP-based CL-ELISA (x) and Immulite kit (y) for detecting Tg showed a good correlation between these two immunoassays (y=1.15 x -0.14, R=0.99). The obtained results open good perspectives for use of SbP/SPTZ/MORPH system in the development of ultra-sensitive immunoassays.