Diammonium salt

Analytical-grade (purity ≥ 98% 99%) TBP (CAS No. 126-73-8) and TCEP 97% (CAS No. 115-96-8), laccase mediator 2,2-azino-bis (3-ethylbenzothriazoline-6-sulphonic acid) diammonium salt (ABTS 98%), 2,6-dymetoxyphenol (DMP, 99%), commercial laccase purified from T. versicolor (20 U/mg), sodium malonate dibasic monohydrate, dimethyl succinate, ammonium acetate, and cytochrome P450 (CYP450) inhibitor 1-aminobenzotriazole (ABT) (98% pure) were purchased from Sigma-Aldrich Co. (Barcelona, Spain). The Microtox bioassay kit was supplied by Strategic Diagnostics Inc. (Newark, NJ, USA). Cyclohexane was purchased from Labkem (Barcelona, Spain). Chromatographic-grade methanol, acetonitrile, and formic acid (≥98%) were obtained from Merck (Darmstadt, Germany). All other chemicals used were analytical grade and acquired from Sigma-Aldrich Co. (Barcelona, Spain).

ABTS (2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Sigma-Aldrich, Warsaw, Poland); acetonitrile (Sigma-Aldrich, Poland); deionized water (Sigma-Aldrich, Poland); ethylacetate (Merck, Warsaw, Poland); methanol (Merck, Warsaw, Poland); ortho-phosphoric acid (Chempur, Warsaw, Poland); phenolic compound standards (purity 99.5–99.9%), including caffeic acid, chlorogenic acid, gallic acid, catechin, epigallocatechin, quercetin, and caffeine (Merck, Poland); and phosphate-buffered saline (Merck, Warsaw, Poland), were used in this study.

The betanin standard (C₂₄H₂₆N2O₁₃), sulfuric acid (H₂SO₄), boric acid (H₃BO₃), formic acid (CH₂O₂), hydrochloric acid (HCL), terephthalic acid (TPA, C₈H₆O₄), ethylenediaminetetraacetic acid (EDTA), 6-hydroxy-2-5-7-8-tetramethylchromo-2-carboxylic acid (Trolox), ascorbic acid (C₆H₈O₆), sodium hydroxide (NaOH), potassium permanganate (KMnO₄), hydrogen peroxide (H₂O₂), potassium sulphate (K₂SO₄), ferrous sulphate (FeSO₄), methyl red, bromocresol green, petroleum ether, anhydrous sodium acetate (CH₃COONa), tetrabutylammonium perchlorate (C₁₆H₃₆N.H₂PO₄), vanillin, tripyridyltriazine (TPTZ, C₁₈H₁₂N₆), iron chloride (FeCl₃), dibasic sodium phosphate (NaH₂PO₄.H₂O), monobasic sodium phosphate (NaH₂PO₄.H₂O), sodium chloride (NaCl), anhydrous monobasic sodium phosphate (Na₂HPO₄), sodium bicarbonate (NaHCO₃) C-211,2,2′-Azobis (2-methylpropionamidine), dihydrochloride (AAPH), 2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS, C₁₈H₂₄N₆O₆S₄), sodium fluorescein (C₂₀H₁₂O₅), potassium hydroxide (KOH), ammonium thiocyanate (NH₄SCN), trichloroacetic acid (Cl₃CCOOH) were purchased from Sigma-Aldrich Chemical Co. (São Paulo, SP, Brazil). Methanol (MeOH), ethanol, acetone, and acetonitrile were purchased from Tedia Company Inc. (Rio de Janeiro, RJ, Brazil). Buthylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), 1,1,3,3-tetramethoxypropane ((CH₃O)₂CHCH₂CH(OCH₃)₂) and 2-thiobarbituric acid (C₄H₄N₂O₂S) were purchased from Sigma-Aldrich Co. HPLC grade Milli-Q water (Merck Millipore, Burlington, MA, USA) was used throughout the experiments.

The following reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA): Folin and Ciocalteu′s phenol reagent, sodium carbonate, 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH), 2,4,6-tris (2-pyridyl)-s-triazine (TPTZ), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) diammonium salt, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), fluorescein sodium salt, potassium persulfate, and ferric sulfate. The following reagents were bought from PanReac AppliChem (Barcelona, Spain): dehydrated sodium phosphate, sodium acetate, ferric chloride, hydrochloric acid, ethanol, formic acid, glacial acetic acid, acetonitrile, and acetone. Sodium hydroxide was purchased from VWR Chemicals (Radnor, PA, USA).
Phenolic standards (degree of purity ≥98%, w/w) were obtained from Extrasynthese (Genay, France) (hydroxytyrosol and oleuropein) and Sigma-Aldrich (St. Louis, MO, USA) (gallic acid, rutin, and caffeic acid).

MaxiSorp microtiter plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with Protein A-affinity purified antibodies at 0.4 µg/ml in 0.1 M carbonate/bicarbonate buffer, pH 9.6. After 2 h blocking with 1.5% (w/v) casein in phosphate-buffered saline with 0.05% Tween 20 (PBST), the microwells were incubated with standards, assay controls and samples diluted in PBST for 1 h at 22°C. A standard curve was obtained using serial 1/2 dilutions from 10–0.04 ng/ml of the D. farinae extract (Allergopharma). House dust extract was used as a positive control, and wheat flour extract was used as a negative control. Each sample was tested at three serial 1/2 dilutions. The captured mite proteins were detected with antigen-affinity purified and biotinylated antibodies (0.1 µg/ml, 1 h, 22°C), followed by streptavidin-peroxidase conjugate (Poly-HRP80-SA, Fitzgerald, Concord, MA, USA) (1/10,000 in PBST, 1 h, 22°C) and finally ABTS substrate solution [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic-acid)] diammonium salt, Sigma-Aldrich, Steinheim, Germany) in 50 mM phosphate-citrate buffer pH 4.2 with 0.015% H₂O₂. The reaction was stopped with 0.32% sodium fluoride, and the absorbance was read at 414 nm. Samples with mite concentrations below the lower limit of detection (LOD) were also analysed using an amplified EIA with QuantaBlu fluorogenic substrate (ThermoScientific, Rockford, IL, USA) instead of ABTS. Sample concentrations were calculated by interpolation of optical density (OD) values or relative fluorescence units on a 4-parameter fitted standard curve using Softmax Pro 5.4.1 (Molecular Devices, Sunnyvale, CA, USA). The LOD was the concentration corresponding to OD  = 0.1 or 600 relative fluorescence units (∼5 times the standard deviation of the background value) above the estimated minimum value (‘parameter A’) of the 4-parameter curve fit function.