Protein A sepharose gel has a high binding capacity toward camel immunoglobulin G (IgG). Ammonium sulfate (NH4)2SO4, monobasic sodium phosphate (NaH2PO4), dibasic sodium phosphate (Na2HPO4), orthophenylenediamine, HRP, bovine serum albumin, and protein A conjugate peroxidase were purchased from Sigma. Sera from camels, immunized goats and rabbits were used as the source of polyclonal camel IgG and anti-camel IgG (Bacterial and parasitic antigens were donated by Prof. Dr. Eman H. Abdel-Rahman).
Ortho phenylenediamine
Manufactured by Merck Group
Sourced in United States, Germany
Ortho-phenylenediamine is a chemical compound used as a laboratory reagent. It is a colorless to pale brown crystalline solid. Ortho-phenylenediamine serves as a precursor for the synthesis of various dyes and pharmaceutical products.
Automatically generated - may contain errors
Lab products found in correlation
96 well plate, by Corning (3 mentions)
Tween 20, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Ovalbumin (ova), by Merck Group (2 mentions)
Ammonium sulfate nh4 2so4, by Merck Group (1 mentions)
Sodium phosphate monobasic nah2po4, by Merck Group (1 mentions)
Sodium phosphate dibasic na2hpo4, by Merck Group (1 mentions)
Multiskan ascent, by Thermo Fisher Scientific (1 mentions)
Igg2b, by Thermo Fisher Scientific (1 mentions)
Pbs t, by Jackson ImmunoResearch (1 mentions)
High binding polystyrene 96 well plates, by Corning (1 mentions)
Peroxidase conjugated rabbit anti human igg, by Jackson ImmunoResearch (1 mentions)
Pbs t, by Merck Group (1 mentions)
Polystyrene plate, by Corning (1 mentions)
Epoch multi volume spectrophotometer system, by Agilent Technologies (1 mentions)
Elisa plate, by Thermo Fisher Scientific (1 mentions)
Elx800uv microplate reader, by Agilent Technologies (1 mentions)
Centrifuge 5804 r, by Eppendorf (1 mentions)
Elisa microplate reader, by Bio-Rad (1 mentions)
39 protocols using ortho phenylenediamine
1
Camel IgG Purification and Detection
2
Cholera Vibrio OSP Antibody ELISA
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Measures of OSP-specific IgG and IgA antibodies in plasma were conducted using enzyme-linked immunosorbent assay (ELISA) as previously described (86 (link)). Briefly, antigens V. cholerae O1 Ogawa OSP with bovine serum albumin (BSA, 1 μg/ml) or V. cholerae O1 Inaba OSP with BSA (1 μg/ml) were prepared in bicarbonate buffer (pH 9.6 to 9.8) and coated on 96-well polystyrene plates. V. cholerae O1 Ogawa and Inaba OSP and BSA conjugates were prepared as previously described using V. cholerae O1 El Tor PIC018 and PIC158 strains, respectively (87 (link)). Plasma was diluted 1:40 in 0.1% BSA with 0.05% Tween in phosphate-buffered saline (PBS) and 100 μl of diluted plasma was plated per well. Following incubation, horseradish peroxidase (HRP)-conjugated secondary antibodies for human IgG and IgA (Jackson ImmunoResearch, West Grove, PA) with a 1:1,000 dilution were applied, and plates were developed using ortho-phenylene diamine (Sigma, St. Louis, MO) in 0.1 M sodium citrate buffer (pH 4.5) and 0.01% hydrogen peroxide. Kinetic measurements were taken at an absorbance of 450 nm for 5 min with 14-s intervals. Values were recorded as milli-absorbance units/min. ELISA data were normalized using the ratio of the optical density of the test sample to the standard of pooled convalescent-phase sera (from previously infected patients with cholera) (88 (link)).
3
Evaluating Murine Antibody Response to MPV
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Two weeks after the last injection of the immunization schedule, a fraction of immunized mice in each group (n = 5) were sacrificed by the overdose of ketamine/xylazine, and their blood was collected by cardiac puncture. An indirect ELISA assay kit (R&D Systems, USA) was employed to determine the MPV-specific serum levels of IgG total, IgG1, and IgG2a antibodies. In brief, 96-well microtiter plates were coated with the MPV in 50 mM carbonate buffer (pH 9.6) overnight at 4 °C. Then, the wells were washed three times with PBS supplied with 0.05% Tween 20. At the next step, the blocking solution (bovine serum albumin 1%) was added to the wells and the plate was incubated for 1 h at 37 °C. After three times wash with PBS, the sera were added (three wells for each sample) and the plate was incubated for another 1 h at 37 °C. HRP-conjugated goat anti-mouse IgG total, IgG1, or IgG2a (Abcam, USA) antibodies were employed as the secondary antibody. Finally, the orthophenylene diamine (Sigma, USA) and 0.15% H2O2 (Sigma, USA) were added and incubated for 30 min to visualize the immune complexes. After stopping the reaction by adding H2SO4, a microplate reader (Bio-Rad 680, USA) was employed to read the absorbance at 490 nm. Complete culture media was used as blank.
4
Enzyme-Linked Immunosorbent Assay for Antibody Characterization
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High-binding, 96-well polystyrene flat bottom plates (Corning, NY, USA) were coated with 15 μg of OVA (Sigma, USA), 1 μg of iIAV or 1 μg of Typhim Vi vaccine per well, each dissolved in 0.1 M carbonate buffer (pH 9.5). Non-specific binding was blocked with 5% non-fat dry milk diluted in PBS pH 7.2. Sera were serially diluted twofold and incubated for 1 h at 37°C. Peroxidase-conjugated anti-mouse IgM, IgG H + L, IgG2a, IgG2b, or IgG3 (Invitrogen, CA, USA) were diluted at a ratio of 1:1,000. The plates were developed with 0.5 mg/mL ortho-phenylenediamine (Sigma, MO, USA) in 0.1 M citrate buffer (pH 5.6) containing 0.08% H2O2 (Sigma, MO, USA). The reaction was stopped with 1.25 M H2SO4, and the optical densities were read at 492 nm using an automatic ELISA plate reader (Multiskan Ascent, Thermo Scientific, Vantaa, Finland). The cutoff value was defined as threefold above the mean values of the negative controls.
High-avidity IgG antibodies were measured including a wash with a mild-denaturing agent to discriminate low-avidity antibodies, which are more likely to dissociate from the antigen–antibody complexes (30 (link)). Briefly, ELISA was performed as described above including a 10 min wash with 7 M urea solution after incubation of sera and before the addition of the secondary antibody.
High-avidity IgG antibodies were measured including a wash with a mild-denaturing agent to discriminate low-avidity antibodies, which are more likely to dissociate from the antigen–antibody complexes (30 (link)). Briefly, ELISA was performed as described above including a 10 min wash with 7 M urea solution after incubation of sera and before the addition of the secondary antibody.
5
Optimized ELISA for IgG Detection
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The conventional ELISA was performed with a microtiter plate coated with the pigeon sera diluted in a carbonate buffer, incubated overnight at 4 °C, and washed 3 times with PBS-Tween 20. Plates were then blocked with PBS-Tween containing 0.5% bovine serum albumin (BSA) for 1 h at 37 °C on a horizontal orbital microplate shaker and washed 3 times with PBS-Tween 20. Patient sera were diluted 1:6250 for the IgG assay in PBS-Tween containing 0.5% BSA. Under these conditions, each sample was studied in triplicate, and plates were incubated for 1 h at 37 °C with shaking. Purified goat anti-human IgG (Fc specific) conjugated with horseradish peroxidase (Calbiochem, Darmstadt, Germany) was added and incubated for 1 h at 37 °C with shaking. Finally, orthophenylenediamine (Sigma Aldrich, Steinheim, Germany) was added and incubated at 37 °C with shaking for 10 min, and the reaction was stopped with H2SO4 1 mol/L. The absorbance was read in a microplate spectrophotometer at 492 nm with a correction factor at 595 nm.
Serum samples were measured simultaneously using conventional ELISA assay versus the detection with the developed method of magnetic beads.
Serum samples were measured simultaneously using conventional ELISA assay versus the detection with the developed method of magnetic beads.
6
ELISA-based Antibody Titer Assay
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Antibody titers against S. Typhi OmpC/F in sera were determined as previously described [22 (link)]. Briefly, high-binding 96-well polystyrene plates (Corning) were coated with 10 μg/ml of the protein preparation in 0.1 M carbonate-bicarbonate buffer, pH 9.5. Plates were incubated for 1 h at 37°C and then overnight at 4°C. Before use, plates were washed three times with PBS containing 0.05% Tween-20 (PBS-T) (Sigma–Aldrich). Non-specific binding was blocked with 5% non-fat dry milk diluted in PBS (PBS-M) for 1 h at 37°C. After washing, sera were diluted 1:40 and stool extracts 1:2, both in PBS-M and twofold serial dilutions were added to the wells. Plates were incubated for 1 h at 37°C, followed by four washes with PBS-T. After 1 h of incubation at 37°C with peroxidase-conjugated rabbit anti-human IgG (1:10,000) or IgM (1:5000) antibody (in PBS-M, Jackson Immuno Research), four washes with PBS-T, ortho-phenylenediamine (0.5 mg/ml; Sigma) in 0.1 M citrate buffer, pH 5.6, containing 0.08% H2O2 was used to develop the reaction. Optical density was read at 492 nm using an automated ELISA plate reader (Tecan). Antibody titers are given as -log2 dilution × 40. Antibody titers were defined as the highest dilution of the sample at which the OD was higher than the mean ± 3 SD of the negative sample values.
7
ELISA for Serum and Mucosal IgG and SIgA
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Serum samples and mucosal washes were tested for the presence of specific IgG and secretory immunoglobulin A (SIgA) using enzyme linked immunosorbent assays (ELISAs). Ninety-six well polystyrene plates (Corning) were coated with 7.5 µg/ml rTgROP17 (100 µl/well) in PBS overnight at 4°C. The plates were washed with PBS containing 0.05% Tween20 (PBST) and blocked for 1 hour at 37°C in PBS containing 5% BSA and then washed with PBS. Serum samples were diluted 1∶200 in PBS, mucosal washes without dilutions were incubated at 4°C overnight in different wells of the coated 96 well plates at 100 µl/well. Next day, the wells were washed and incubated with 100 µl of HRP-labelled goat anti-mouse antibody (AbD Serotec; diluted 1∶2500 in PBS) for serum specific IgG for 1 h at 37°C. The wells were washed extensively, incubated with orthophenylene diamine (Sigma, USA) and 0.15% H2O2 for 30 min, and enzyme reactions were terminated by the addition of 50 µl of 1 M H2SO4. Optical density (OD) was measured in an ELISA plate reader (Epoch Multi-Volume Spectrophotometer System, Biotek, USA) at 492 nm. All samples were analysed in triplicate for at least three independent experiments.
8
Quantification of T. gondii Antibodies
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We performed ELISAs to determine T. gondii-specific serum antibody levels. In short, the 96-well plates (Costar, USA) were coated with STAg (10 μg/well) and incubated at 4 °C overnight. The next day, plates were washed three times with ELISA solution and blocked with PBS containing 1% bovine serum albumin (BSA) for 1 h at 37 °C. Next, the plates were washed and incubated with the mouse sera diluted in PBS (1:100) for 1 h at 37 °C. After washing, plates were incubated with horseradish peroxidase-conjugated anti-mouse IgG (diluted 1:4,000 in PBS with 1% BSA) for 1 h at 37 °C. After washing, orthophenylenediamine (Sigma, USA) and 0.15% H2O2 were added to the plates. The plates were then incubated in the dark for 30 min at 37 °C, and the reaction was terminated by adding 2 M H2SO4. The OD was detected at 405 nm with an ELISA reader (ELX800, USA). All of the samples were run four times.
9
Detecting Anti-T. gondii Antibodies
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Anti-T. gondii IgG, IgG1, and IgG2a antibodies were detected using enzyme-linked immunosorbent assay (ELISA) in accordance with the manufacturer’s instructions (Southern Biotech Co., Ltd, Birmingham, USA). Briefly, microtiter 96-well plates were coated overnight at 4 °C with pET30-toxofilin protein antigen (diluted in 0.1 M carbonate buffer (pH 9.5) for optimal binding. The plates were washed three times with PBS containing 0.05% Tween20 (PBST) and then blocked with 1% bovine serum albumin for 1 h at 37 °C with gentle shaking. After three washes with PBST, the plates were incubated with mouse sera diluted 1:100 in PBS for 1 h, followed by incubation with goat anti-mouse IgG, IgG1, or IgG2a secondary antibodies for 1 h to detect the target antibody and isotype control, respectively. Finally, the immune enzymatic reaction complexes were visualized by incubating with ortho-phenylenediamine (Sigma, USA) and 0.15% H2O2 for 30 min. Reactions were stopped by adding 2 M H2SO4, and the results were read at OD450 nm using an ELISA plate reader. An average of three independent ELISAs for each serum sample was recorded.
10
Induced Anti-Ovalbumin Immune Response
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To induce an anti-ovalbumin immune response, mice were immunized with 50 μl of 50 mg/ml OVA with complete Freund's adjuvant in the left footpads on day 0 and day 7. Mice were sacrificed on day 21 and serum was collected. To determine the anti-OVA IgG response, ELISA plates (Nunc Maxisorp, Thermo Scientific) were coated with 5 μg/ml ovalbumin (Sigma-Aldrich, Budapest) overnight in PBS, followed by saturation with 0.1% gelatine in PBA-0.1% Tween-20 (Sigma–Aldrich, Budapest, Hungary) for 1 h at 37°C. After saturation and washing in PBS-Tween diluted sera of mice were added to blocked plates, followed by washing. Next, horseradish peroxidase conjugated rabbit anti-mouse polyclonal antibody was added, and after incubation peroxidase activity was detected using ortho-phenylenediamine and H2O2 (Sigma-Aldrich, Budapest, Hungary) in citrate-phosphate buffer, pH: 5.0. The reaction was stopped with 4 M H2SO4. Samples were measured at 492 nm in duplicates.
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