All activity assays were performed in duplicate or triplicate, on 96-well microtiter plates, and analyzed with a microplate reader. Peroxidase activity, with 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma, Oakville, ON, Canada), was measured as described previously [26 (link)]. Briefly, 10 μL of sample was combined with 80 μL 0.75 mM H2O2 (Sigma) and 110 μL TMB solution (2.9 mM TMB in 14.5% DMSO (Sigma), and 150 mM sodium phosphate buffer at pH 5.4), and the plate was incubated at 37 °C for 5 min. The reaction was stopped by adding 50 μL of 2 M H2SO4 (Sigma), and absorption was measured at 450 nm, to estimate MPO activity [41 (link),93 (link)].
2 m h2so4
Manufactured by Merck Group
Sourced in United States
2 M H2SO4 is a concentrated sulfuric acid solution with a molarity of 2 moles per liter. It is a commonly used laboratory reagent for various applications in research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (5 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
96 well elisa plate, by Greiner (2 mentions)
Tmb substrate, by Abcam (2 mentions)
Spectramax abs plus spectrophotometer, by Molecular Devices (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
L 15 media, by Thermo Fisher Scientific (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Sodium carbonate, by Merck Group (1 mentions)
Multiskan skyhigh photometer, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Goat anti chicken iga horseradish peroxidase conjugate, by Thermo Fisher Scientific (1 mentions)
Pbs tween, by Merck Group (1 mentions)
Endo grade ovalbumin protein, by Merck Group (1 mentions)
Tetramethylbenzidine tmb, by BD (1 mentions)
Nunc cell culture, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Tmb substrate kit, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg hrp conjugate, by Thermo Fisher Scientific (1 mentions)
11 protocols using 2 m h2so4
1
Quantitative Peroxidase Activity Assay
2
Quantifying Neutrophil Infiltration in Livers
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To quantify neutrophil (granulocyte) infiltration livers were harvested, washed in cold HBSS and homogenized in fresh cold HBSS using a gentleMACS™ dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). The homogenates were frozen at -80°C, thawed and centrifuged at 3000 × g for 15 min. Myeloperoxidase (MPO) activity was assessed from the supernatants with 3,3′,5,5′-tetramethylbenzidine (TMB from Sigma Aldrich, St Louis, MO, USA) (34 (link)). Briefly, 10 µl sample were combined with 80 µl 0.75 mM H2O2 and 110 µl TMB solution (2.9 mM TMB in 14.5% DMSO and 150 mM sodium phosphate buffer at pH 5.4), and the plate was incubated at 37°C for 5 min. The reaction was stopped by adding 50 µl 2 M H2SO4 (Sigma), and absorption was measured at 450 nm to estimate MPO activity. To obtain values per organ the total volume of each liver suspension was measured. Values of livers from uninfected control animals were subtracted as background prior to plotting of the data.
3
SAA Quantification in Stimulated RTS-11 Cells
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Supernatants (100 μL) from RTS-11 IL-1β stimulation and control 4, 6, 8, 10, 12, and 24 h post-stimulation were used. An amount of 100 μL of L-15 media (Gibco™, Thermo Fisher Scientific, Waltham, MA, USA) (background), along with 0.2, 0.5, 1, 5 and 10 ng recombinant SAA (rSAA) diluted in L-15 media, served as positive control and was used for the calibration curve. Samples were added to 96-well ELISA plates (Greiner Bio-One, Kremsmünster, Austria) and incubated at 4 °C overnight. Wells were blocked with 300 μL of 1% BSA (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 1 h at RT. The anti-SAA biotin-linked antibody was diluted in 1% BSA + 0.05% Tween (Sigma-Aldrich, St. Louis, MO, USA) and 100 μL (75 ng/well) was added to each well. After 1 h incubation at RT, wells were washed three times with 300 μL PBS + 0.05% Tween (PBST). A total of 100 μL of poly-HRP (1:20,000 in PBST) was added, and the plate was incubated at RT for 30 min. The plate was washed five times with PBST followed by the addition of 50 μL TMB substrate (Abcam, Cambridge, UK) for 30 min at RT. The reaction was stopped with 50 μL 2 M H2SO4 (Sigma-Aldrich, St. Louis, MO, USA), after which the plate was read at 450 nm using a SpectraMax® ABS Plus spectrophotometer (Molecular Devices, San Jose, CA, USA).
4
Enzyme-Linked Immunosorbent Assay Protocol
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Flat-bottomed 96-well plates (LPS solution, Daejeon, Republic of Korea) were coated with 0.6% sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) and 0.3% sodium carbonate (Sigma-Aldrich, St. Louis, MO, USA) in DW and incubated overnight at 4 °C. After being washed with PBS containing 0.1% Tween 20 (TPBS), the plates were blocked with 5% skim milk (LPS solution, Daejeon, Republic of Korea) in PBS for 4 h at room temperature. The wells were washed with TPBS, 80 μg protein samples were added to each well, and the plates were incubated overnight at 4 °C with shaking. The plates were then washed and the appropriate detection antibody was added (Table S2 ) and incubated at 4 °C overnight with shaking. After being rinsed with TPBS, HRP-conjugated antibody solution was added (Table S2 ) and the plates were incubated at room temperature for 2 h. After being washed, the HRP substrate solution (3,3′,5,5′-tetramethylbenzidine; Sigma-Aldrich, St. Louis, MO, USA) was added and the plates were incubated for 15 min at room temperature in the dark to allow for color development. The reaction was stopped with an equal volume of stop solution (2 M H2SO4; Sigma-Aldrich, St. Louis, MO, USA) and absorbance at 450 nm was measured using a microplate reader (Multiskan SkyHigh Photometer; Thermo Fisher Scientific, Rockford, IL, USA).
5
Antibody Quantification in Chicken Sera and Secretions
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The level of antibodies against C.jejuni protein in intestinal secretions and sera of chicken was quantified by ELISA.
Ninety-six-well plates (Nunc, Thermo Scientific) were coated overnight at 4 °C with whole-cell C. jejuni proteins (20 μg/well), washed three times with PBS containing 0.02 % Tween 20 (Sigma-Aldrich), blocked for 1 h at 37 °C with PBS containing 2 % bovine serum albumin (Sigma-Aldrich), washed as described previously, and incubated for 1.5 h at room temperature with either diluted sera (1:300) or intestinal secretion samples (1:10). The plates were developed with 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich), using goat anti-chicken IgA horseradish peroxidase conjugate (Thermo Scientific, Rockford, IL) or rabbit anti-chicken IgY (whole molecule)-peroxidase (Sigma-Aldrich) (dilution 1:2000), respectively. The plates were incubated with the substrate for 25 min at room temperature and then the colorimetric reaction was stopped by adding 2-M H2SO4 (Sigma-Aldrich). Absorbance was measured at 490 nm using an ELISA reader (Tekan). Each sample was analyzed in duplicate.
Ninety-six-well plates (Nunc, Thermo Scientific) were coated overnight at 4 °C with whole-cell C. jejuni proteins (20 μg/well), washed three times with PBS containing 0.02 % Tween 20 (Sigma-Aldrich), blocked for 1 h at 37 °C with PBS containing 2 % bovine serum albumin (Sigma-Aldrich), washed as described previously, and incubated for 1.5 h at room temperature with either diluted sera (1:300) or intestinal secretion samples (1:10). The plates were developed with 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich), using goat anti-chicken IgA horseradish peroxidase conjugate (Thermo Scientific, Rockford, IL) or rabbit anti-chicken IgY (whole molecule)-peroxidase (Sigma-Aldrich) (dilution 1:2000), respectively. The plates were incubated with the substrate for 25 min at room temperature and then the colorimetric reaction was stopped by adding 2-M H2SO4 (Sigma-Aldrich). Absorbance was measured at 490 nm using an ELISA reader (Tekan). Each sample was analyzed in duplicate.
6
Quantifying Antigen-Specific Serum IgG Levels
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For detection of antigen-specific serum IgG, microtitre plates (Maxisorp, Nunc) were incubated overnight with either 20 μg/mL Endo Grade® Ovalbumin protein (Sigma-Aldrich), 10 μg/mL HA protein, or 2 μg/mL RBD protein in bicarbonate buffer. 10% FBS was applied to prevent non-specific binding, prior to application of serially diluted serum samples. A final incubation was performed with a variety of Ig isotypes; IgGTOTAL (G21040), IgG1 (A10551), IgG2a/c (PA129288), IgG2b (M32407), IgG3 (M32707) conjugated to HRP (Life Technologies). Tetramethylbenzidine (TMB; BD Biosciences) and 2M H2SO4 (Sigma-Aldrich) was applied to develop and stop reactions, before immediately being read at 450 nm. Wash steps were performed 5x between each incubation with 10% PBS-Tween (Sigma-Aldrich).
7
Quantification of Recombinant Atlantic Salmon SAA
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Recombinant Atlantic salmon SAA protein was diluted in L-15 media (Gibco™, Thermo Fisher Scientific, Waltham, MA, USA). An amount of 100 μL of L-15 media (background control), along with 0.2, 0.5, 1, 5 and 10 ng rSAA diluted in L-15 media, were added to 96-well ELISA plates (Greiner Bio-One, Kremsmünster, Austria) and incubated at 4 °C overnight. Wells were blocked with 300 μL of 1% BSA (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 1 h at RT. The anti-SAA biotin-linked antibody was diluted in 1% BSA + 0.05% Tween (Sigma-Aldrich, St. Louis, MO, USA) and 100 μL (75 ng/well) was added to each well. After 1 h incubation at RT, wells were washed three times with 300 μL PBS + 0.05% Tween (PBST). A total of 100 μL of poly-HRP (1:20,000 in PBST) was added, and the plate was incubated at RT for 30 min. The plate was washed five times with PBST, followed by the addition of 50 μL TMB substrate (Abcam, Cambridge, UK) for 30 min at RT. The reaction was stopped with 50 μL 2 M H2SO4 (Sigma-Aldrich, St. Louis, MO, USA), after which the plate was read at 450 nm using a SpectraMax® ABS Plus spectrophotometer (Molecular Devices, San Jose, CA, USA).
8
Myeloperoxidase Activity Assay for Cornea Samples
9
Quantitative ELISA for RBD/HA Antibodies
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One hundred microliters of RBD or influenza A H1N1 HA recombinant protein (4 μg/ml; A42579, Thermo Fisher Scientific) was added into micro titer plates (Nunc Cell Culture, Thermo Fisher Scientific) for overnight at 4°C. After that, 200 μl of 5% (w/v) bovine serum albumin (B6717, Sigma-Aldrich) in phosphate-buffered saline with 0.1% Tween 20 (PBS-T) buffer was added to each well to block nonspecific binding and incubated at 37°C for 1 hour. After three times of wash with PBS-T, serum samples with serial dilutions (1:100, 1:1000, 1:3000, 1:5000, 1:10,000, 1:50,000, 1:100,000, and 1:200,000) and control serum samples (1:100 dilution) were added to the wells and incubated at 37°C for 1.5 hours. After washing intensively, 100 μl of horseradish peroxidase–labeled anti-mouse IgG secondary antibody with 1:10,000 dilution was added for 1 hour of incubation at 37°C. After three times of wash, 100 μl of trimethylboron was added into each well and incubated at room temperature for 30 min. Fifty microliters of 2 M H2SO4 (Sigma-Aldrich) was used for stopping the reaction, and the optical absorption was determined at 450 nm via a plate reader. The endpoint IgG titer was determined as the highest reciprocal dilution of serum that exhibits an optical density greater than twofold of the mean control group.
10
Brucella O-PS Antibody ELISA Assay
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107 CFU/mL bacteria were harvested, resuspended in 0.5% phenol (Sigma Aldrich Rehovot, Israel) saline and incubated for 2 days at 37 °C. After 2 days, deactivated bacteria were centrifuged at 4500 rpm for 10 min and dissolved in 5 mL of coating buffer as previously described [70 (link)]. Two-hundred microliters of the dissolved bacteria were then placed in wells of a 96 well ELISA plate (Thermo-Nunc Maxisorp), incubated overnight at 37 °C, washed thrice with PBS-Tween and blocked with 1.5% Bovine Serum Albumin (Sigma) for 2 h at room temperature. The plate was then washed thrice with PBS-Tween and 100 µL of purified anti-O-PS Murine Monoclonal antibody (SVANOVIR® Brucella-Ab C-ELISA kit; 1:10) was added to each well and incubated for 2 h at 37 °C. The plate was washed again, incubated for 1 h at 37 °C with 100 µL of goat anti-mouse IgG HRP conjugate (Thermo Fisher Scientific, Waltham, MA, USA; 1:1200), and developed with TMB (Tetramethylbenzidine) substrate (TMB Substrate Kit, Thermo Fisher Scientific) for 5 min. To stop the reaction, 100 µL of 2 M H2SO4 (Sigma Aldrich) was added to each well and absorbance was measured at 450 nm in a BioTek microplate reader. The experiment was repeated twice, independently, in triplicate. All the work with Brucella strains was performed at a biosafety level 3 laboratory in the Kimron Veterinary Institute, Bet Dagan, Israel.
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