ИСТИНА

Enzyme immunoassay is one of the widely used bioanalytical methods. Depending on the substrate used, the products of its oxidation can be detected spectrophotometrically or by chemiluminescence (CL). The latter method is based on the enzymatic oxidation of luminol resulted in an electronically excited 3-aminophthalate dianion, which emits light as it returns to the ground state. Horseradish peroxidase (HRP) is one of the most popular enzyme labels. However, because of poor activity towards luminol, compounds known as enhancers have to be added to the substrate mixture to increase CL intensity. Opposite, previously isolated and characterized in our laboratory anionic tobacco peroxidase (TOP) was extremely active towards luminol even in the absence of chemiluminescence enhancers. Also, the protocol for the production of recombinant wild-type TOP in active form from E.coli inclusion bodies was developed. To reactivate the insoluble enzyme a special refolding procedure was required. In this work we optimized the conditions for refolding of wild-type TOP resulted in an increased yield up to 80%. Obtained recombinant TOP was used to produce conjugates with antibodies (TOP-IgG). To compare TOP-IgG with commonly used in practice HRP-conjugated IgG the quantitative sandwich enzyme immunoassay was performed. CL reaction conditions were optimal for each enzyme. It was shown, that use of TOP as enzyme label provides greater sensitivity of the assay.