ИСТИНА

Chemiluminescence immunoassay (CLIA) provides a sensitive detection of antigens, haptens or antibodies. This assay is based on the enzymatic oxidation of luminol resulted in light emission and widely used when analyte concentration is at low level. CLIA does not require long incubations or the addition of stopping reagents, as is the case in most colorimetric assays. The most commonly used labeling enzymes are horseradish peroxidase and alkaline phosphatase. However, because of poor activity towards luminol, compounds known as enhancers have to be added to the substrate mixture to increase chemiluminescence (CL) intensity. The development of enzymes and substrates with higher activity, better stability and more suitable kinetic curve is the hotspot in CLIA research. Previously isolated and characterized in our laboratory anionic tobacco peroxidase (TOP) was extremely active towards luminol even in the absence of CL enhancers [1]. In addition, TOP exhibited a higher stability to inactivation with H2O2 [2]. Also a procedure for heterologous expression of recombinant enzyme (rTOP) in E. coli strain was developed [3]. In this work we have conjugated rTOP and rabbit anti-mouse IgG using SATA (N-succinimidyl S-acetylthioacetate) as a crosslinking reagent. To compare rTOP-IgG with commonly used in practice HRP-conjugated IgG the quantitative sandwich enzyme immunoassay was performed. Chemiluminescence reaction conditions were optimal for each enzyme. The intensity of chemiluminescence in case of rTOP-IgG was about two orders of magnitude higher in comparison to HRP-IgG. Likewise, the slope of calibration curve for rTOP-IgG was significantly bigger. Thus, using of rTOP as enzyme provides better sensitivity and detection precision in CLIA. However, commercial application of rTOP as enzyme label requires a high product yield. Nevertheless, previously reported conditions for rTOP production did not provide sufficient amount of enzyme for commercial manufacture. Therefore, we also performed optimization of the conditions for higher rTOP yield. Since rTOP was accumulated in insoluble form in cytoplasmic E. coli inclusion bodies, the in vitro refolding procedure was required to reactivate the enzyme. It includes four main steps: (1) isolation of inclusion bodies; (2) solubilization in 6M urea; (3) dilution of solubilized enzyme in refolding medium; (4) concentration and purification of rTOP. The refolding step is a major bottleneck in described procedure as it involves the formation of native disulfide bridges and incorporation of heme and Ca2+ ions. Extensive screening experiments were performed to optimize the refolding conditions (such as pH value, protein concentration, and concentration of redox reagents, hemin, urea, and glycerol). Optimized refolding system was characterized by higher yields and minimum product losses in the purification step. This resulted in an increased active enzyme yield by up to 83%.