Laccase (EC 1.10.3.2) activity was measured in triplicate at each sampling date from planktonic (varying from 3 to 8 mg of fungal dry mycelium) and biofilm (one square of leaf/wood) samples using the 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) substrate (Sigma-Aldrich, St. Louis, MO, United States). Enzyme activity assays were conducted according to the protocol of Johannes and Majcherczyk (2000) (link) with some modifications. Substrate saturating conditions were fixed at 3 mM ABTS and incubations were performed during 1 h at 20°C, under agitation (80 rpm) in the dark. ABTS transformation was measured spectrophotometrically (420 nm) using an Ultrospec 2000 device (Pharmacia Biotech, Trowbridge, United Kingdom). The enzymatic activity was expressed as 1 U = 1 μmol ABTS oxidized/g mycelium dry mass/h (ε420 = 36 M-1 cm-1, Johannes and Majcherczyk, 2000 (link)). Oven dry mass (DM) was determined systematically for each sample and used to correct laccase activity (activity/h/g DM).
Abts substrate
Manufactured by Merck Group
Sourced in United States, Germany
ABTS substrate is a chromogenic substrate used in various biochemical and immunological assays. It functions as an electron donor, undergoing a color change reaction when oxidized by enzyme-catalyzed reactions. The resulting colored product can be measured spectrophotometrically, allowing for quantitative analysis of the enzymatic activity or analyte concentration in the sample.
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Lab products found in correlation
Cell death detection elisa, by Merck Group (3 mentions)
Anti mpo antibody, by Bio-Rad (2 mentions)
Goat anti chicken igg antibodies coupled to horseradish peroxidase, by Fortis Life Sciences (2 mentions)
Multiskan go, by Thermo Fisher Scientific (2 mentions)
Immulon 2, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Sandwich elisa kit, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Horseradish peroxidase, by Fortis Life Sciences (1 mentions)
96 well flat bottom plate, by Thermo Fisher Scientific (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Synergy 4 hybrid microplate reader, by Agilent Technologies (1 mentions)
Skanit software, by Thermo Fisher Scientific (1 mentions)
Nunc immuno microwell 96, by Thermo Fisher Scientific (1 mentions)
Spectrostar nano microplate reader, by BMG Labtech (1 mentions)
Flat bottom tissue culture plates, by Sarstedt (1 mentions)
Affinipure goat anti mouse igg igm h l, by Jackson ImmunoResearch (1 mentions)
Human il 6 elisa kit, by Thermo Fisher Scientific (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
18 protocols using abts substrate
1
Laccase Activity Quantification in Fungal Samples
2
ELISA for HBV Antibody Detection
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Polystyrene microtiter plates (Immulon 2, Thermo Fisher Scientific, 3455) were coated with either C-HBV, recombinant HBsAg or S1/S2-Core fusion protein diluted to a final concentration of 1.0 µg/mL in coating buffer (15 mM Na2CO3, 35 mM NaHCO3 pH 9.6). A final volume of 100 μL was added to each well and plates were incubated at 4°C for 16 h. Plates were washed twice with Tris Buffered Saline + 0.005% Tween 20 (Sigma-Aldrich, SRE0031) and blocked with blocking buffer (PBS containing 2% BSA) at room temperature for 1 h. Serum samples were serially diluted in blocking buffer, added into corresponding wells and incubated for 2 h at room temperature. Antibodies were detected with rabbit anti-sheep IgG (H + L) HRP labeled (Kirkegaard & Perry Laboratories, 5260–0057) diluted 1:3000. ELISAs were developed using 150 µg/mL ABTS substrate (Sigma-Aldrich, 10102946001) diluted in 0.055 M citric acid (pH 4.0) with 0.3% H2O2. The reaction was stopped after 30 min by the addition of 10% SDS. Optical density (OD) of the reaction product was measured at λ 405 nm using a λ 490 nm reference on a Microplate reader (Bio-Rad Laboratories, 1681000). A positive titer was defined as the reciprocal of the final serum dilution for which an OD reading was obtained that exceeded the mean + 2SD of the reading obtained with a negative serum sample.
3
ELISA for MPO-DNA Complexes
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An in-house ELISA was used to quantify MPO-DNA complexes. Briefly, after overnight coating with anti-MPO antibody (2 μg/mL; 0400-0002, Bio-Rad) at 4°C, a 96-well plate was blocked with 2.5% BSA in PBS for 2 hours at room temperature. The plate was subsequently washed before incubation for 90 minutes at room temperature with 20% human or mouse plasma in blocking buffer. The plate was washed 5 times, and then incubated for 90 minutes at room temperature with anti-DNA antibody (1:10; Cell Death Detection ELISA, 11544675001, Sigma-Aldrich). After 5 washes, the plate was developed with ABTS substrate (10102946001, Sigma-Aldrich).
4
Sandwich ELISA for Cytokine Quantification
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Sandwich CD54 (ICAM-1) and IL-6 ELISAs: HUVEC cells were seeded into 24 well-plates and treated immediately with tested extracts for 30 minutes. MeOH/Tris was used as a vehicle for controls and TNFα (10 ng/ml) as inflammation stimuli. After 24 hours of treatment, culture medium was removed and collected into microfuge tube. Concentrations of cytokines (IL-6, ICAM-1) produced by cells into the medium was assessed using sandwich ELISA kits (PeproTech, Rocky Hill, NJ, USA). 96 well-plates for ELISA (Nunc, Thermo Fisher Scientific, Rockford, USA) were coated with capture antibody. After overnight incubation, plates were washed with 0.05% Tween in PBS and blocked with 1% BSA in PBS (Sigma-Aldrich, Munich, Germany) for 1 hour. Subsequently, plates were washed with PBS with Tween (0.05%) and standard or sample was added for 2 h. After washing, detection antibody was applied for another 2 hours. Avidin-HRP conjugate was incubated for 30 minutes and thereafter HRP activity was estimated by ABTS substrate (Sigma-Aldrich, Munich, Germany) and absorbance was measured at OD405nm in a multimode plate reader (Infinite 200 PRO, Tecan Group, Männedorf, Switzerland).
5
ELISA-Based Serum Anti-NP Antibody Analysis
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Serum anti-NP antibody levels were measured by ELISA as described below. 96-well flat-bottom plates (Thermo Fisher Scientific) were coated with 10 μg/ml NP54-BSA (Biosearch Technologies), and then the wells were blocked with 5-fold diluted Blocking-One (Nacalai Tesque). Appropriately diluted serum samples were added to the wells and incubated at room temperature for 2 h. The plates were washed and incubated for another 1 h with diluted anti-IgM, -IgG1, -IgG2c and -IgG2b antibodies conjugated with horse radish peroxidase (Bethyl Laboratories), respectively. Bound secondary antibody was assessed by incubation with ABTS substrate (Sigma-Aldrich). Absorbance at 405 nm (A405) in each well was measured.
For the analysis of affinity maturation, appropriately diluted serum samples were assayed using an NP4-BSA (Biosearch Technologys) -coated 96-well plate and an NP54-BSA-coated plate. The A405(NP4)/A405(NP54) ratio was calculated as relative affinity.
For the analysis of affinity maturation, appropriately diluted serum samples were assayed using an NP4-BSA (Biosearch Technologys) -coated 96-well plate and an NP54-BSA-coated plate. The A405(NP4)/A405(NP54) ratio was calculated as relative affinity.
6
Human GM-CSF Sandwich ELISA
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Sandwich ELISA for human GM-CSF was performed with the Human GM-CSF Mini ABTS ELISA Development Kit (Peprotech) along with ABTS substrate (Sigma-Aldrich) according to the manufacturer’s instructions.
7
Quantification of Serum Immunoglobulins
8
Quantification of MPO-DNA Complexes in Peritoneal Fluid
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An in-house ELISA was used to quantify MPO–DNA complexes in peritoneal fluid from sham and cecal slurry treated seven to 10-day-old mice, 24 h after injection, as previously described.24 Briefly, after overnight coating with anti-MPO capture antibody (2 µg/mL; 0400–0002, Bio-Rad) at 4 °C, a 96-well plate was blocked with 2.5% bovine serum albumin in PBS for 2 h at room temperature. The plate was subsequently washed, before incubating for 90 min at room temperature with 10% peritoneal fluid in blocking buffer. The plate was washed five times, and then incubated for 90 min at room temperature with anti-DNA detection antibody (1:20; Cell Death detection ELISA, 11544675001, Sigma). After five washes, the plate was developed with ABTS substrate (Sigma).
9
ELISA for Specific Antibody Responses
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ELISA assays for Abs against the pathogen Ags was carried out as previously described.7 (link) Briefly, round-bottom 96-well ELISA microtiter plates (Costar) were coated at 4 ° C overnight with 1–3 μg/ml KLH or OVA-conjugated M2e or SP70 peptide (Mimotopes) for specific Ag Ab responses, or with 1.5 μg/ml rat GL117 mAb for anti-rat Ig Ab responses. Unbound peptide or protein was washed away (PBS, 0.05% Tween 20) and serially diluted serum samples (PBS, 5% milk powder) were plated and incubated at 4 ° C overnight. Bound mouse IgG Abs was detected using donkey anti-mouse IgG-HRP (Millipore, catalogue # AP192P) and visualised using ABTS substrate (Sigma). Optical density (OD) was measured at 405–490 nm. End point titres were calculated by using cut-off values defined as at least five times the OD of a no-serum negative control. To determine the level of Ig isotypes, bound mouse IgG1 (catalogue # 1070-05), IgG2a (catalogue # 1080-05), IgG2b (catalogue # 1090-05), IgG3 (catalogue # 1100-05) and IgM (catalogue # 1021-05) were detected using goat anti-mouse isotype Ig HRP (SouthernBiotech).
10
Enzyme-Linked Immunosorbent Assay for Art v 1-Specific Antibodies
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ELISA was performed to determine Art v 1-specific Ig in the sera of the differently immunized mice. ELISA plates (NUNC maxisorb, Sigma Chemicals) were coated with 2 μg/ml of recombinant Art v 1 protein in carbonate buffer (pH 9.6) overnight. Plates were washed with PBS plus 0.05% Tween (PT) two times and blocked with PT plus 1% BSA at room temperature for 2 h. Sera of immunized mice were diluted in PT plus 0.5% BSA 1:10 for IgE, 1:500 for IgG2 and 1:2000 for IgG1 determinations, respectively. Plates were incubated with 50 μl of diluted sera at 4 °C overnight and subsequently washed five times with PT. Specific Ig were detected by addition of 100 μl of monoclonal rat-anti-mouse Ig-subclass specific antibodies (BD Pharmingen, Palo Alto, CA) diluted 1:500 in PT 0.5% BSA at 37 °C for 3 h. After 5 washes with PT 100 μl of 1:2000 diluted goat-anti-rat horseradish peroxidase coupled antibody (GE Healthcare) were added and incubated at 37 °C for 30 min and at 4 °C for 30 min. Finally, plates were washed 5 times with PT and incubated with 100 μl ABTS substrate (Sigma Chemicals, St. Louis, MO). Plates were developed at room temperature in the dark, and extinction (optical density 405 nm) was measured with an ELISA reader (Multiskan GO) equipped with the SkanIt software (ThermoFisher Scientific, Fremont, CA) after 1 h.
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