2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid abts

Escherichia coli DH5α competent cells were used for cloning plasmids and grown in Luria–Bertani (LB) medium. Pichia pastoris X33 was used for the production of heterologous protein. YPD, BMGY, and BMMY media were prepared according to the manual of the Pichia Expression Kit.
HMF, DFF, HMFCA, FFCA, and FDCA were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., China. 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was purchased from Sigma‒Aldrich Co., Ltd., USA.

The reagents used were ethanol (CTR, Monterrey, MX), methanol (Meyer, Monterrey, MX), 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) (Sigma-aldrich, St. Louis, MO, USA), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (Sigma-aldrich, Oakville, CA), Folin-Ciocalteu’s phenol (Sigma-aldrich, St. Louis, MO, USA), trolox 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Sigma-aldrich, Steinheim, DE), gallic acid (Sigma-aldrich, Hong Kong, CN).

Folin-Ciocalteu (FCR), sodium carbonate (Na₂CO₃),2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) ABTS, α-Tocopherol (Vitamin E), 2,6-ditert- butyl-4-methylphenol (BHT), gallic acid, quercetin, and AlCl₃ were purchased from Sigma (Sigma-Aldrich, Germany). All the organic solvents and other chemicals used in the present study were of analytical grade and were obtained from Sigma-Aldrich (Sigma Aldrich, Germany).

2,2-Diphenyl-1-picrylhydrazyl (DPPH) was obtained from the Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Potassium peroxodisulfate was obtained from SHOWA Chemical Co., Ltd. (Chuo-ku, Japan).

All 16 amino acids used in the experiments were provided by Neocrema Co., Ltd. (Seoul, Republic of Korea). Also, taurine, theanine, and GSH were provided by NeoCrema. Ascorbic acid, 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

A standard rutin sample, a standard vitamin C (Vc) sample, and a standard tea polyphenol sample were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). NaOH, NaNO₂, AlNO₃, ethanol, phosphoric acid, and concentrated hydrochloric acid were obtained from Sinopharm Chemical Reagents Co. Ltd. (Shanghai, China). Standard Escherichia coli strain C83902, Staphylococcus aureus strain ATCC 25923, and Bacillus subtilis strain WL2 were obtained from the China Institute of Veterinary Drug Control (Beijing, China).

Standard protocol for ELISA was used as described in our previous studies (25 (link),26 (link)). In brief, a 96-well microtiter plate (LLC; Immunochemistry Technologies, Bloomington, MN, USA) was coated overnight at 4°C with recombinant NPM1 protein at a final concentration of 0.5 μg/ml in phosphate-buffered saline (PBS). The antigen-coated wells were blocked with gelatin post-coating solution at room temperature for 2 h. Human sera diluted at 1:100 with were added to the antigen-coated wells and incubated for 2 h at room temperature, washed and then incubated with HRP-conjugated goat anti-human IgG (Caltag Laboratories, San Francisco, CA, USA) at a 1:4,000 dilution. The substrate 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS; Sigma-Aldrich, St. Louis, MO, USA) was used to detect the immune complexes. The average optical density (OD) value at a wavelength of 405 nm was used for data analysis. The cut-off value designating a positive reaction was the mean optical density of 90 normal human sera plus 3 standard deviations (SD).