Enzyme linked immunosorbent assay kit

Urinary samples were centrifuged at 4000 rpm, and then each sample was assessed for 8-OHDG using an enzyme-linked immunosorbent assay kit from Cusabio Biotech. All the reagents were of analytical grade. Concentrations of 8-OHDG were estimated by a spectrophotometer and calculated by measuring the optical density of 575 nm. Renal cortex MDA levels were quantified using the lipid peroxidation MDA assay kit (Beyotime Institute of Biotechnology, Jiangsu, China), according to the manufacturer's protocol and as described in our previous report [16 (link)].

To test the effect of toxoflavin on trichothecene biosynthesis, mRNA levels of the trichothecene biosynthesis genes, Tri5 (FGSG_03537, trichodiene synthase) and Tri6 (FGSG_03536, transcription factor for trichothecene synthesis), as well as trichothecene production, were analysed. Briefly, each strain was incubated in 50 ml GYEP (10 g of glucose, 1 g of yeast extract, and 1 g of peptone in 1 l)^{35 (link)} supplemented with or without toxoflavin (80 mg l⁻¹) at 25 °C on a rotary shaker (200 r.p.m.) for 5 d. Total RNA was purified from the mycelia using a NucleoSpin RNA Plant Kit (MACHEREY-NAGEL GmbH & Co. KG), according to the manufacturer’s protocol. The amounts of Tri5 and Tri6 transcripts were quantified by qRT-PCR with specific primers for each gene^{60 (link)}. Concentrations of DON in the culture filtrates were quantified with an enzyme-linked immunosorbent assay kit (CUSABIO, College Park, MD, USA), according to the manufacturer’s instructions^{61 (link)}.

One day after WYHZTL formula and XAV-939 administration, the animals were sacrificed. Collecting blood sample and standing to clot for 2 h at room temperature. Then centrifugation at 1000×g for 10 min, and obtain upper serum carefully to assay immediately. ELISA analysis was performed using enzyme-linked immunosorbent assay kit (Cusabio Technology, China) on a SpectraMax iD3 microplate reader instrument (Molecular Devices, USA). Reagents, and samples or standards were prepared, and experiments were performed according to the manufacturer’s instructions. Briefly, standard or sample were added to each well separately, and incubated at 37 °C for 2 h. Then standards or samples were removed without washing. Add Biotin-antibody to each well, and incubate at 37 °C for 1 h. Remove liquid of each well and wash. Add HRP-avidin to each well, and incubate at 37 °C for 1 h. Remove liquid of each well and wash. Add TMB Substrate to each well, and incubate at 37 °C for 20 min. Add Stop Solution to each well, and immediately detect the optical density of each well by a SpectraMax iD3 microplate reader instrument set to 450 nm. At last, the concentration of target protein was calculated according to standard curve. Each experiment was performed three times independently.

A commercial kit (Jiancheng Institute of Biotechnology, Nanjing, China) was used to measure the concentration of malondialdehyde (MDA) in the ventral midbrain according to the manufacturer’s instructions. Similarly, levels of 3-nitrotyrosine (3-NT) and 8-hydroxy-2-deoxyguanosine (8-OHdG) were determined using an enzyme-linked immunosorbent assay kit (CUSABIO BIOTECH CO., Ltd., Wuhan, China) by following the manufacturer’s instructions.