2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts)

To test laccase production in liquid cultures, PDB was supplemented with 150 μM of CuSO₄ and 0.2 mM of ABTS. A dark‐purple color appeared after 5−7 days of incubation and served as an indicator of laccase activity. According to the literature data (Gramms, 2017 (link)), the first step of ABTS oxidation leads to the formation of green (cyan) cation radical (ABTS^•+) predominantly present in the initial phase of this process. However, prolonged incubation with laccases leads to its further oxidation to form a purple product (i.e., ABTS²⁺).
Likewise, the secretion of laccases was also detected on plates by supplementing potato dextrose agar (PDA) with 150 μM of CuSO₄ and 0.2 mM ABTS (VWR Chemicals) or guaiacol (Merck). Additional assays were carried out in 1.5 ml Eppendorf tubes to confirm the presence of laccase activities in protein preparations used for zymography. These assays were performed by mixing 980 μl of laccase reaction buffer (100 mM sodium acetate buffer, pH 4.5; 0.2 mM ABTS) with 20 μl of protein samples.

To measure serum total IgA, ninety-six-well microtiter plates were coated with anti-IgA mAb (Absea Biotechnology Ltd., Beijing, China) in carbonate-bicarbonate buffer, pH 9.6, overnight at 4°C. The plates were washed three times with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST) and then blocked with 200 μL/well of 2% bovine serum albumin (BSA) for 2 h at room temperature. The serum samples were diluted 1 : 6000, added, and incubated for 1 h at room temperature. Then, the plates were washed and horse radish peroxidase- (HRP-) conjugated anti-human IgA mAb (Absea Biotechnology Ltd., Beijing, China) was added and incubated for 1 h at room temperature. After washing, the color was developed using ABTS (AMRESCO, Solon, USA) as a substrate and the absorbance was measured at 405 nm with a microplate reader (BioTek Synergy 4).
To measure serum IgA1, anti-IgA mAb (Absea Biotechnology Ltd., Beijing, China) was coated. After washing, the serum samples were diluted 1 : 20000, added, and incubated for 1 h at room temperature. Then, the plates were washed and anti-IgA1 mAb (AbD Serotec, Kidlington, UK) was added in 1 : 1000 dilution and incubated for 1 h at room temperature. The color was developed using ABTS as a substrate.