Uv transparent plate

The oxidation of EGCG was followed by UV–Vis spectroscopy. Solutions of EGCG were prepared as described above. In total, 10 mM EGCG solutions were dissolved in 10 mM sodium phosphate buffer pH 7.4, corresponding to the conditions under which a stock solution of EGCG ${}_{ox}$ for insulin experiments was produced, and in 20 mM citric acid, pH 7, corresponding to the conditions under which a stock solution of EGCG ${}_{ox}$ for the $\alpha$ -synuclein experiments was produced. The oxidation of EGCG was carried out by incubating the solutions at 60 ${}^{\circ }$ C for 0–22 h. Subsequently, the solutions were diluted and the spectra were recorded in the wavelength range between 250 nm and 500 nm in a UV-transparent plate (Corning, Corning, NY, USA, number 3679) using a Spark (Tecan, Mannedorf, Switzerland) microplate reader. To monitor the stability of EGCG under the used aggregation experiments, 172 $\mathsf{\mu }$ M EGCG in 100 mM NaCl, 100 mM sodium phosphate buffer, pH 2.4, 172 $\mathsf{\mu }$ M EGCG in 20% acetic acid, and 100 mM NaCl were incubated at 60 ${}^{\circ }$ C for 0–22h, corresponding to the conditions of the insulin aggregation experiments, and 125 $\mathsf{\mu }$ M EGCG in 150 mM citric acid at pH 6 and pH 7 were incubated at 37 ${}^{\circ }$ C for 0–22 h, corresponding to the conditions of the $\alpha$ -synuclein aggregation experiments.

ELISA plates (Greiner Bio-One) were coated overnight at 4°C with 100 µl of 5 µg/ml rabbit anti-ADA2 polyclonal antibodies in PBS with 0.02% NaN₃. After washing the plates 3 times with 200 µl PBS-Tween 20 buffer and blocking with 2% BSA in PBS with 0.02% NaN₃ for 1 hour, 100 µl of recombinant ADA2 standards diluted in PBS containing 10% FBS or serum samples diluted in PBS containing 0.02% NaN₃ were added to the wells. The plates were incubated for 1 hour at RT on a shaker. Subsequently, the plates were washed 3 times with 200 µl PBS-Tween 20, and 100 µl of 2 mM adenosine in 20 mM Tris-HCl pH 6.8, 10 μM ZnCl₂, 0.02% NaN₃ was added. The plate was incubated at 37°C for 16-24 hours. The reaction was stopped by transferring 20 µl of the reaction mixture into a UV-transparent plate (Corning) containing 180 µl of water per well. The ratio of absorbance at 265 and 245 nm was detected on a Thermo Fischer Multiscan Go Reader. The ADA2 concentration in the samples was determined from a standard curve generated with the recombinant protein.

ELISA plates (Greiner Bio-One) were coated overnight at 4°C with 100 µl of 2 µg/ml Streptavidin (New England BioLabs) in PBS with 0.02% NaN₃. After washing the plates 3 times with 200 µl PBS-Tween 20 buffer and blocking with 2% BSA in PBS with 0.02% NaN₃ for 1 hour, 100 µl of 1 µg/ml biotinylated rabbit anti-ADA1 polyclonal antibodies (Abcam) in 10% FBS with 0.02% NaN₃ were added to the wells. The plate was incubated for 30 min at room temperature on a shaker. After washing the plates 3 times with 200 µl PBS-Tween 20, 100 µl of recombinant ADA1 standards diluted in PBS containing 0.5% BSA or saliva samples diluted in PBS with 0.02% NaN₃ were added to the wells. The plates were incubated for 1 hour at RT on a shaker. Subsequently, the plates were washed 3 times with 200 µl PBS-Tween 20, and 100 µl of 0.3 mM adenosine in 20 mM Tris-HCl pH 7.3, 10 μM ZnCl₂, 0.02% NaN₃ was added. The plate was incubated at 37°C for 18-24 hours. The reaction was stopped by transferring 80 µl of the reaction mixture into a UV-transparent plate (Corning) containing 120 µl of water per well. The ratio of absorbance at 265 and 245 nm was detected on a Thermo Fischer Multiscan Go Reader. The ADA1 concentration in the samples was determined from a standard curve generated with the recombinant protein.