Levels of secretory IgA were determined by enzyme linked immunosorbent assay (ELISA) in small bowel intestinal fluids. Microtitre plates (Nunc-Immuno Plates, MaxiSorp) were coated with goat anti-mouse antibody (Southern Biotechnology, Birmingham, AL, USA) in carbonate-bicarbonate buffer (0.1 M Na2CO3/NaHCO3—pH 9.6) for 18 h at 4 °C. Wells were washed with washing solution (saline 0.9% plus 0.05% tween 20) and blocked with 200 µL of 0.05% casein in PBS for 1 h at room temperature. Intestinal fluids previously centrifuged at 432g for 20 min were added to the plate and diluted in PBS-0.25% casein (two times until dilution 1:80). After incubation of 1 h at room temperature, plate was washed and biotin conjugated anti-mouse IgA antibody (Southern Biotechnology) in PBS-0.25% casein (1:10.000) was added to the wells. After incubation of 1 h at 37 °C, peroxidase-streptavidin goat anti-mouse IgA (Southern Biotechnology, Birmingham, AL, USA) was added; plate was incubated for 1 h more and, then, coated with 100 µL/well of orthophenylenediamine (OPD) (1 mg/mL) (Sigma, St. Louis, MO, USA) and 0.04% H2O2 substrates. Color was developed at room temperature and reaction was stopped by the addition of 20 µL/well of 2 N H2SO4. Absorbance was measured at 492 nm using a Bio-Rad Model 450 Microplate Reader. Results were expressed as concentration (µg/mL), according to the standard curve.
Ortho phenylenediamine opd
Manufactured by Merck Group
Sourced in United States
Ortho-phenylenediamine (OPD) is a chemical compound used as a laboratory reagent. It is a colorless crystalline solid. OPD is commonly used in various analytical and diagnostic applications, particularly in colorimetric assays and enzyme-linked immunosorbent assays (ELISA).
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Lab products found in correlation
Model 450 microplate reader, by Bio-Rad (3 mentions)
Biotin conjugated anti mouse iga antibody, by Southern Biotech (2 mentions)
Tween 20, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Sepharose cl 4b, by GE Healthcare (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Costar high binding plates, by Corning (1 mentions)
Profinity epoxide resin, by Bio-Rad (1 mentions)
Goat anti mouse iga hrp, by Southern Biotech (1 mentions)
Anti rabbit hrp for caspase 1, by Merck Group (1 mentions)
Elisa plate, by Corning (1 mentions)
Hrp conjugated anti human igg, by Merck Group (1 mentions)
Anti iga antibodies, by Southern Biotech (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Cetyltrimethylammonium bromide, by Merck Group (1 mentions)
Gelatin type a, by Merck Group (1 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by Merck Group (1 mentions)
Hyaluronic acid, by Merck Group (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Coomassie brilliant blue r 250, by Merck Group (1 mentions)
10 protocols using ortho phenylenediamine opd
1
Measurement of Intestinal Secretory IgA
2
Protein Purification and Characterization Techniques
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Materials and reagents used in these experiments included bicinchoninic acid (Thermo Fisher, Rockford, IL, USA), diethylamine (Fluka, Buchs, Switzerland), ELISA 96-well microplates (Maxi Sorp, 442,404; Nunc, Roskilde, DK), Costar high-binding plates (9018; Corning, NY, USA), High-trap Protein G HR and Sepharose CL-4B (both from GE-Healthcare Life Sciences, Chicago, IL, USA), Profinity epoxide resin (Bio-Rad, Madrid, Spain), Tween-20 and ortho-phenylenediamine (OPD) (Sigma, St. Louis, MO, USA). Buffers were PBS (phosphate-buffered saline, pH 7.4), PBST (PBS with 0.05% Tween-20), PBSM blocking buffer (5% non-fat dry milk in PBS with 10 μg/L NaN3), and 0.1 M glycine-hydrochloride pH 2.5.
3
Phage-Induced Systemic Immune Response in Poultry
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To assess whether the phages were able to produce a systemic immune response, ELISA was performed at 42-day-old birds as described (Santos et al. 2018 (link)), with modifications. The microtitration plates were sensibilised with 1 μg/well of phage M13 diluted in 50 mM bicarbonate buffer (pH 8.6) for 16 h at 4 °C. After three washes with PBS-T (PBS + Tween 20 at 0.05%), the plates were blocked with PBS-T with 5% skim powdered milk for 1 h at 37 °C and subsequently washed three times with PBS-T, followed by incubation with different bird serum at different dilutions (1:50 and 1:100) at 37 °C for 1 h. The controls were performed without phages or serum. After five washes with PBS-T, the anti-IgY was added, followed by incubation at 37 °C for 1 h. The wells were washed five times, and the ligation antigen/antibody was detected by the addition of ortho-phenylenediamine (OPD) at 1 mg/mL with 3% H2O2 (Sigma Chemical Co.). The reaction was stopped by the addition of 4 N sulfuric acid. Reactivity was determined in a plate reader (Titertek Multiskan Plus, Flow Laboratories, USA) at a wavelength of 492 nm. The results represented the absorbance in wells sensitised with phage subtracted from that of the control wells (not sensitised with phages).
4
Intestinal sIgA Quantification by ELISA
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Levels of sIgA in the intestinal fluid were determined by enzyme linked immunosorbent assay (ELISA) as previously described [23 (link)]. Briefly, microtitre plates (NUNC, Thermo Scientific, Waltham, MA, USA) were coated with goat anti-mouse UNLB antibody (Southern Biotechnology, Birmingham, AL, USA) in coating buffer (pH 9.8) overnight at 4°C. Wells were then washed with a saline 0.05% tween solution and blocked with 200 μL of PBS with 0.05% (w/v) casein for 1 h at room temperature. The supernatants obtained from the intestinal fluids after centrifugation at 432 g for 20 min at 4°C and the diluted standards in PBS-0.25% casein (1:10) were then added to the plate and incubated for 1 hour at 37°C. After washing, goat anti-mouse IgA HRP (Southern Biotechnology, Birmingham, AL, USA) was added and plates were incubated for 1 h at 37°C. The colour reaction was developed at room temperature with the addition of 100 μL/well of orthophenylenediamine (OPD) (1 mg/mL) (Sigma, St. Louis, MO, USA), 0.04% H2O2 substrate in sodium citrate buffer. The reaction was finally stopped by the addition of 20 μL/well of 2 N H2SO4. Absorbance was measured at 492 nm using a Bio-Rad Model 450 Microplate Reader. Results were expressed as concentration (μg/mL), according to the standard curve.
5
Quantifying Caspase Levels in Plasma
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Plasma caspase levels were measured using a double antibody sandwich ELISA. Ninety-six well plates were coated with unlabeled polyclonal antibodies to caspase-1 or -3 (Santa Cruz Biotechnology, USA) in 50 μl of antibody solution (20 μg/ml in PBS) and incubated for 2 h at room temperature (RT). After incubation, the wells were washed with PBS-Tween-20 washing buffer, and 100 μl blocking buffer (3% BSA / PBS + 0.02% sodium azide) was added to all of the wells and incubated for 2 h at RT. This step was followed by another washing step and addition of the sample (50 μl), which was incubated at RT for 2 h. The plates were washed, and 50 μl of the polyclonal antibody to Caspase-1 or -3 was added to the respective plates. After a 2-h incubation at RT, a horseradish peroxidase (HRP)-labeled antibody was added to the wells (anti-rabbit HRP for Caspase-1 and anti-goat HRP for Caspase-3) (Sigma, USA). The plates were again incubated for 2 h at RT and washed at the end of incubation. Ortho phenylene diamine (OPD, Sigma, USA) was used as a substrate. The anti-human Caspase-1 used was non-cross reactive with Caspase-1 p10 or Caspase-1 p20. It did not detect Caspase-1 of mouse or rat origin. The anti-human Caspase-3 used was known to react with the carboxy terminal prodomain of caspase-8 (also designated as pro Mch5, MACH alpha 1 or FLICE) of human origin.
6
ELISA for Serum Protein Antigens
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ELISA with rSeCP (rSeCP-ELISA) and ES antigens (ES-ELISA) were performed as previously described [27 (link)]. Briefly, the ELISA plates (Corning, USA) were coated with rSeCP (2.5 μg/ml) and ES proteins (2.5 μg/ml) in 100 μl of bicarbonate buffer (pH 9.6) at 4°C overnight. After blocking with 5% skim milk in PBST at 37°C for 1 h, the following reagents were sequentially added and incubated at 37°C for 1 h as follows: (1) sera diluted at 1:100 in PBST, and (2) HRP-conjugated anti-human IgG (Sigma, USA) diluted at 1: 5 000. After the final wash, the reactions were detected by the substrate ortho-phenylene diamine (OPD, Sigma, USA) plus H2O2 and stopped with 50 μl/well of 2 M H2SO4. Optical density (OD) values at 490 nm were determined with a microplate reader (TECAN, Austria). All samples were run in duplicate. Test sera/negative serum OD values <2.1 were regarded as negative and those ≥2.1 as positive. The cut-off values of rSeCP-ELISA and ES-ELISA were 0.34 and 0.42, respectively.
7
Plasma Sialidase and CTB-Specific Antibody Responses
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Plasma sialidase and CTB-specific IgA, IgG, and IgM responses were assessed using modification of enzyme-linked immunosorbent assay (ELISA) protocols (35 (link), 36 (link)). Briefly, 96-well polystyrene plates (Nunc F, Denmark) were coated with sialidase (2.5 μg/ml) and then blocked with 1% bovine serum albumin (BSA; Sigma, St. Louis, MO) in phosphate-buffered saline (PBS). We then added a 1:25 dilution of plasma (100 μl/well), followed by horseradish peroxidase-conjugated anti-human immunoglobulin antibodies of the relevant isotype (1:1,000 dilution; Jackson ImmunoResearch, West Grove, PA). We detected bound antibody with ortho-phenylenediamine (OPD; Sigma, St. Louis, MO) in 0.1 M sodium citrate buffer (pH 4.5) and 0.012% hydrogen peroxide. Optical density was measured at 450 nm for 5 min, and the rate of change in optical density was measured as milli-absorbance units per minute. The absorbance value of each sample was then normalized and expressed as ELISA units by calculating the ratio of the sample to a standard of pooled plasma. A positive response was defined as 1.5-fold increase in ELISA units from day 2 to convalescence (either day 7 or day 30).
8
Quantification of Secretory IgA in Intestinal Fluids
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Levels of secretory IgA (sIgA) were determined by enzyme-linked immunosorbent assay (ELISA) in small bowel intestinal fluids (Carvalho et al., 2017a (link)). Microtiter plates (Nunc-Immuno Plates, MaxiSorp) were coated with anti-IgA antibodies (Southern Biotechnology, Birmingham, AL, United States) for 18 h at 4°C. The plates were washed with saline (NaCl 0.9%) added with Tween 20 (0.05%) and blocked with 200 μl PBS-casein (0.05%) for 1 h at room temperature. Intestinal fluid samples were diluted in PBS-casein (0.25%) and then added to the plate. After incubation for 1 h at room temperature, the wells were washed and biotin-conjugated anti-mouse IgA antibody (Southern Biotechnology) diluted in PBS-casein (0.25%) (1: 10,000). The plates were incubated for 1 h at 37°C and anti-IgA conjugated to streptavidin peroxidases (1:10,000) were added (Southern Biotechnology). After 1 h of incubation, 100 μl of orthophenylenediamine (OPD) (Sigma, St. Louis, MO, United States) and H2O2 (0.04%) were added to each well. Plates were kept away from light until the coloration developed. The reaction was stopped by addition of 2 N H2SO4. Reading was performed on a plate reader (Bio-Rad Model 450 Microplate Reader) at 492 nm absorbance. The results were measured in concentration of sIgA (μg) per ml of intestinal fluid, according to the standard curve.
9
Reagents for Biochemical Assays and Protein Analysis
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Bovine serum albumin (BSA), gelatin type A, 1,10-phenanthroline (PHE), ethylene diamine tetracetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), cetyltrimethylammonium bromide (CTAB), Coomassie Brilliant Blue R-250, Triton X-100, Tween 20, hyaluronic acid, Concanavalin A (Con A) from Canavalia ensiformis, Wheat germ agglutinin from Triticum vulgaris (WGA), 3, 3”-diaminobenzidine tetrahydrochloride (DAB) and ortho-phenylenediamine (OPD) were purchased from Sigma (Missouri, USA). Goat anti-horse (GAH) IgG labeled with alkaline phosphatase (IgG-AP) or with horseradish peroxidase (IgG-HRPO), 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), nitroblue tetrazolium (NBT) and BCA assay kit were purchased from Promega (Wisconsin, USA). Brij-35 P was purchased from Fluka—BioChemika (Werdenberg, Switzerland). EnzChek Phospholipase A2 Assay Kit was purchased from Invitrogen (California, USA). Fluorescent Resonance Energy Transfer (FRET) substrate, Abz-RPPGFSPFRQ-EDDnp, was synthesized and purified as described [20 (link)].
10
Quantifying Humoral Immune Response to Vaccination
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Effects of vaccination on humoral response levels were assessed by measuring total antigen-specific IgG, IgG1 and IgG2a using sera collected on days 0, 20, 41 and three weeks after the final immunization (day 62) using ELISA. Briefly, STAg of the T. gondii RH strain was diluted to 10 μg/mL with coating buffer (pH 9.6) and divided into Costar 96-well flat-bottom microtiter plates (Thermo Fisher Scientific, USA) using 100 μL per well. After an overnight incubation at 4°C, 100 μL of blocking buffer (PBST containing 2.5% of skim milk) was added to each well to block non-specific binding sites and plates were incubated at 37°C for 1 hr. Then, plates were washed thrice with PBS buffer containing 0.05% of Tween-20 (PBST). Then, 100 µL of diluted mouse sera (1:100 dilution) was added to each well and incubated at 37°C for 1 hr. After washing the plates, 100 µL of 1:750 diluted horseradish peroxidase-conjugated goat anti-mouse IgG, IgG1 or IgG2a (Abcam abID#97265, 97240, 97245, UK) was added to each well and then plates were incubated at 37°C for 1 hr. After incubation and wash steps, substrate of ortho-phenylenediamine (OPD) (Sigma-Aldrich, USA) was added to the wells. Reactions were stopped using 2M H2SO4 solution and the optical density (OD) was measured using automated ELISA reader (Model LX800; Biotek, Winooski, USA) at 490 nm. All samples were tested in triplicate.
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