Elisa

Viral RNA was extracted (EZ1 Advanced XL-QIAGEN; Hilden, Germany) and analysed using a quantitative one-step real-time RT-PCR with primers and probes targeting the S segment. The Rotor-Gene Probe RT-PCR kit (QIAGEN, Hilden, Germany) and Rotor-Gene Q platform were used. Viral load was measured with the intra-assay standards as described before [19 ].
The presence of IgG and IgM antibodies against CCHFV in serum was detected using an indirect enzyme-linked immunosorbent assay (ELISA) (Vector-Best, Novosibirsk, Russia). ELISA tests were completed in two sessions. According to the manufacturer’s instructions, optical densities (OD) > 0.214 and > 0.250 for IgM and IgG, respectively, were considered as positive results in the first session and > 0,214 and > 0,240 for the second. Higher OD values that could not be read by the ELISA reader were taken as 3.0. All tests were performed at the virology laboratory of the Public Health Institute of Turkey.

Levels of IFN-γ, IL-10, IL-1β and IL-6 in the culture supernatants were determined by ELISA (Vector-Best, Russia). Lower limit of detection were 2.0 pg/ml for IFN-γ, 1.0 pg/ml for IL-10 and IL-1β, 0.5 pg/ml for IL-6. Levels of IL-17 were determined by ELISA kits from eBioscience (USA). Lower limit of detection was 0.5 pg/ml. In all cases of ELISAs, the instructions of the kit manufacturers were followed. Data are expressed as pg/ml or as the percentage of cytokine production by stimulated cells in the absence of GA.

The serum concentrations of IL-6, TNF-α and VEGF were determined by ELISA (Vector Best, Russia) using DS2 automated ELISA analyzer (Dynex Technologies, USA), according to the manufacturer’s instructions.

Prior to mixing with target cells (Nalm6-CD20), CAR T cells had their TexMACS medium replaced with IMDM. CAR-T cells were then co-cultured overnight with target cells at a 1:2 E:T ratio in IMDM medium containing 10% FBS without exogenous cytokines, in duplicate. The next day, cell cultures were centrifuged at 500× g and supernatants were transferred into new plates. Levels of human IFN-γ and IL-2 were analyzed by ELISA according to the manufacturer’s protocols (Vector Best, Russia).

The performance of the newly developed immunosensor was tested for the determination of immunoglobulins against TBEV in two immunological products: human immunoglobulins against TBEV (FSUC SIC "Microgen", Moscow, Russia) with concentrations not less than 80 and 160 IU·mL⁻¹. The electrochemical measurements were carried out as described in Section 2.6.
The ELISA (Vector-Best, Novosibirsk, Russia) was selected as a comparative method, where the detection is based on the indirect assay. The amount of the bound conjugate (horseradish peroxidase-labelled antibodies to TBEV) with human immunoglobulin against TBEV is determined by colour reaction using a peroxidase substrate-hydrogen peroxide and 3,3′,5,5′-tetramethylbenzidine. The intensity of staining is proportional to the concentration of antibodies to TBEV in the immunological product.