The humoral immune response of immunized animals was evaluated by measuring mucosal anti-ESAT-6 sIgA levels in macerated mouse colons. For this, colons were macerated in buffer solution containing anti-proteases in a ratio of 1 ml of solution to 100 mg of tissue. The IKA T10 Basic Homogenizer workcenter (IKA® Brasil, Campinas, Brazil) was employed for this purpose. The homogenates were centrifuged, the supernatant was collected and the measurement of sIgA was performed by indirect ELISA as follows. Recombinant ESAT-6 (5 μg/ml) was used to coat microliter plates overnight (Pereira et al., 2015 (link)). Then, samples without dilution were added and incubated for 1 h. Next, horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies (Sigma-Aldrich, Darmstadt, Germany) diluted in PBS/casein (1/8000) were added and incubated for 2 h. Finally, orthophenylenediamine (OPD−1 mg/ml; Sigma Aldrich, Darmstadt, Germany) was used for color development as an indicator. Absorbance was measured at 492 nm using an ELISA Expert Plus Microplate Reader (Biochrom Asys, Cambridge, United Kingdom).
Horseradish peroxidase hrp conjugated goat anti mouse antibody
Manufactured by Merck Group
Sourced in United States
Horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody is a laboratory reagent used in various immunoassays and detection methods. It consists of a goat-derived antibody that specifically binds to mouse antibodies, conjugated with the enzyme horseradish peroxidase. The HRP enzyme can catalyze a colorimetric or chemiluminescent reaction, enabling the detection and visualization of target mouse antibodies in a sample.
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Lab products found in correlation
Streptavidin hrp conjugate, by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Coomassie brilliant blue, by Bio-Rad (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Mouse monoclonal anti flag antibody, by Merck Group (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence kit, by Beyotime (1 mentions)
Tetramethylbenzidine substrate, by Thermo Fisher Scientific (1 mentions)
6 protocols using horseradish peroxidase hrp conjugated goat anti mouse antibody
1
Mucosal Anti-ESAT-6 sIgA Quantification
2
Dot Blot and Western Blot Analysis of Recombinant HCV Core Protein
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For dot blot analysis of proteins, equal concentration of TSP (5 µg-25 µg) from plant extracts, 5 µg from eHCV as positive control and 25 µg of non-agroinfiltrated plant extract were directly dotted on a nitrocellulose membrane. The membrane was dried at room temperature, blocked with 5% (w/v) skim milk in PBS-Tween (1/1000 v/v), and after three washing steps incubated with anti-C/N terminal 5xHis antibody (Qiagen) 1:10000 in TBS-T for two hours at room temperature. Following the washing steps, the membrane was incubated with 1:8000 diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies (Sigma, USA) for one hour. Finally, 3-3’diaminobenzidine (DAB, Sigma, USA) was used for color development.
For SDS-PAGE and western blotting analyses, purified HCV core from E. coli and plant and plant crude extracts of the recombinant HCV core protein expressed by P19 co-agroinfiltrated pBI121 and PVX vectors were loaded onto a 12% SDS-polyacrylamide gel; by the end of electrophoresis, the protein bands were either stained with coomassie brilliant blue (Bio-Rad) or transferred to the PVDF membrane. Subsequently, the corresponding protein bands on the membrane were identified by biotinylated anti-core polyclonal antibody (Abcam, UK) (1:1000 dilution) and streptavidin HRP conjugate (Sigma, USA) (1:4000 dilution), respectively, and were detected by TMB as substrate.
For SDS-PAGE and western blotting analyses, purified HCV core from E. coli and plant and plant crude extracts of the recombinant HCV core protein expressed by P19 co-agroinfiltrated pBI121 and PVX vectors were loaded onto a 12% SDS-polyacrylamide gel; by the end of electrophoresis, the protein bands were either stained with coomassie brilliant blue (Bio-Rad) or transferred to the PVDF membrane. Subsequently, the corresponding protein bands on the membrane were identified by biotinylated anti-core polyclonal antibody (Abcam, UK) (1:1000 dilution) and streptavidin HRP conjugate (Sigma, USA) (1:4000 dilution), respectively, and were detected by TMB as substrate.
3
SARS-CoV-2 Focus Forming Assay
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One day prior to inoculation, 24-well cell culture plates were seeded with 5x104 Vero-81 cells. Virus stocks were serially diluted 10-fold then added to cells (after growth media was removed) for one hour at 37°C. After incubation, cells were overlaid with 1% methylcellulose in OptiMEM I (Gibco) supplemented with 2% FBS, nonessential amino acids and 100U/ml penicillin, 100μg/ml streptomycin and 0.25μg/ml Amphotericin B, and incubated at 32°C. After four days incubation, overlay was removed, cells were washed with phosphate-buffered saline (PBS) and fixed in 80% methanol. Cells were blocked in 5% non-fat dried milk (blocking buffer) then incubated with anti-prM MAb 2H2 and anti-E MAb 4G2 diluted in blocking buffer. Cells were washed with PBS, then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Sigma) diluted in blocking buffer. Plates were washed and foci were developed using TrueBlue HRP substrate (KPL).
4
Analyzing VP4 Protein Expression in Bacterial Mutants
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In an effort to analyse the expression of VP4 protein in mutant bacterial strains, VP4/△Alr W56, VP4/△Alr HLJ-27, and VP4/W56 were inoculated in a corresponding MRS medium at 37°C without shaking, and the bacterial precipitation was acquired by centrifugation at 12,000 rpm for 2 min. After lysis with lysozyme, ultrasonic breaking, and centrifugation, the proteins in the supernatants were detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in a 15% gel, followed by a western blot assay. The proteins were further electrotransferred onto polyvinylidene fluoride membranes (Millipore, Milford, MA, USA). Next, the proteins were incubated with mouse anti-flag monoclonal antibody (Sigma, St. Louis, MO, USA) at a dilution of 1:1500 and then with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Sigma, St. Louis, MO, USA) as a secondary antibody with a dilution of 1:8000. The colour development of immunoblots was carried out using an ECL reagent (Thermo Scientific, Durham, NC, USA).
5
Recombinant Virus Detection by Western Blot
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The rescued recombinant virus rBEV-E0 was also detected by western blot according to standard procedures. In brief, MDBK cells were infected with the 10th generation recombinant virus and parental virus, and incubated for 12 h. Viral samples were loaded on a 12% polyacrylamide gel, transferred to nitrocellulose membranes, and then blocked with 5% skim milk in Tris-buffered saline with Tween 20 for 2 h at 37 °C. The membranes were then incubated with anti-BJ101-VP1 monoclonal antibody (mAb) 4B2 and anti-BVDV E0 polyclonal antibody (PcAb, 1:3000; both prepared in our laboratory) at 37 °C for 1 h followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (1:4000; Sigma-Aldrich). Detection was performed using an enhanced chemiluminescence kit (Beyotime, Shanghai, China).
6
Influenza HA Receptor Binding Assay
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We tested the receptor binding specificity of the HA protein for α2,3- or α2,6-linked sialic acid using a solid-phase binding assay with two different glycopolymers: α-2,6 glycans (6′SLN, Neu5Aca2-6Galb1-4GlcNAcb-PAA-biotin) and α-2,3 glycans (3′SLN, Neu5Aca2-3Galb1-4GlcNAcb-PAA-biotin), as previously described (Imai et al., 2012 (link)). Briefly, a streptavidin-coated high-binding capacity 96-well plate (Pierce) was incubated with PBS containing different concentrations (starting from 2.4 μM) of biotinylated glycans at 4°C overnight. After the glycan solution was removed, the plates were washed four times with ice-cold PBS and then incubated at 4°C overnight with PBS containing 128 HA units of purified influenza virus. After washing, the plates were incubated for 4 h at 4°C with mouse antibody against influenza NP. The plates were then washed four times and incubated with horseradish peroxidase (HRP)-conjugated goat-anti-mouse antibody (Sigma-Aldrich) for 2 h at 4°C. After four washes, the plates were finally incubated with tetramethylbenzidine substrate (Thermo Scientific), and the reaction was stopped with 50 μl of 2 M H2SO4. Absorbance was determined at 450 nm.
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