The specificity of the produced anti-S. typhimurium and anti-S. enteritidis IgYs were also evaluated using Western blot analysis. The formalin-inactivated immunogens were electrophoresed on SDS-PAGE polyacrylamide gel using 12% resolving and 5% stacking gels; protein bands were transferred to nitrocellulose membrane using semi-dry blotting. Non-specific binding sites on the blotting membranes were blocked with sterile PBS containing 3% w/v skimmed milk and incubated at 4 °C overnight; then, the membranes were washed 3 times with PBST. Blotting membranes were incubated individually with either anti-S. typhimurium or anti-S. enteritidis IgYs diluted 20 times with blocking buffer for 1 h at 37 °C. The membranes were then washed 3 times with PBST to remove the excess antibody and incubated with horseradish peroxidase-conjugated rabbit anti-chicken IgG (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:10,000 for 30 min at 37 °C. Finally, the membranes were washed 3 additional times and binding of specific IgYs to the corresponding bacteria was visualized by staining the membranes with 0.05% diaminobenzidine (DAB) (Sigma Aldrich, St Louis, MO, USA) in 50 mM Tris pH 7.4 containing 0.05% H2O2.
Horseradish peroxidase conjugated rabbit anti chicken igg
Manufactured by Merck Group
Sourced in United States, Sao Tome and Principe
Horseradish peroxidase-conjugated rabbit anti-chicken IgG is a laboratory reagent used in various immunoassay techniques. It consists of rabbit-derived antibodies specific to chicken immunoglobulin G (IgG) that are conjugated to the enzyme horseradish peroxidase. This conjugate can be used to detect and quantify the presence of chicken IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
5 protocols using horseradish peroxidase conjugated rabbit anti chicken igg
1
Specificity of Anti-Salmonella IgYs
2
ELISA Assay for Chicken IgY Antibody Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In brief, wells of Microtiter plates were coated with 100 μl of antigen solution appropriately diluted with 0.05 M carbonate buffer (pH 9.6). After overnight incubation at 4°C, the plates were washed, and 200 μ1 of PBS (pH 7.4) containing bovine serum albumin (1% in PBS) was added to the wells to block the uncoated surface. After being blocked, each well was washed three times with 200 μL of PBS-Tween (PBS-T; 0.85% NaCl - 0.01 M phosphate buffer, pH 7.2) (containing 0.05% Tween 20), and IgY from immunized hens at different time intervals was applied to the well in triplicate for reaction with the antigen for 2 h at 37°C. After each well was washed again with 200 μL of PBS-T, 100 μL of horseradish peroxidase-conjugated rabbit anti-chicken IgG (Sigma Chemical Co.) diluted (1:1000) with PBS-T was added to each well, and the plate was incubated at 37°C for 2 h. Each well was washed again with 200 μL of PBS-T, and then 100 μL of TMB solution with H2O2. The reaction was stopped after 20 min with 4N H2SO4 (50 μl per well), and the intensity of color developed was measured at 490 nm with a microplate reader. IgY anti-TBC titer was defined as the maximum dilution multiple of the sample with an OD. Crude IgY from nonimmunized hens was used as the control.
3
Quantification of Serum Antibody Titers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An enzyme-linked immunosorbent assay (ELISA) was conducted to check the serum antibody titer. The 96-well microtiter plates (Gibco, Grand Island, NY, United States) were coated overnight at 4°C with PBS (pH 7.4), containing 1 μg/mL of the purified CSBV antigen. CSBV-coated plates were washed three times with 0.05% Tween-20 in PBS (PBST). The wells were blocked with 300 μL of PBS containing 3% skim milk for 1 h at 37°C. The wells were washed again three times with PBST. Next, 100 μL of appropriately diluted serum (1:1000 dilution) preparations from immunized and non-immunized hens at different time intervals were added to the wells. The plates were then incubated at 37°C for 1 h, and washed three times with PBST, before adding horseradish peroxidase-conjugated rabbit anti-chicken IgG (Sigma Chemical Co., St. Louis, MO, United States) diluted (1:5,000) in PBS, and incubating for 45 min at 37°C. 3,3′,5,5′-Tetramethylbenzidine substrate solution was added, and plates were incubated for 15 min, before sulfuric acid was added to stop the reaction. The optical density (OD) was measured on an ELISA plate reader, using a 450-nm filter, and the ODs were statistically analyzed using statistical software SPSS17.
4
Incorporation of RVG and RVGTM into NDV Virions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BHK-21 cells were incubated with rL, rL-RVG, or rL-RVGTM at an MOI of 5.0, and cell sediments were prepared for analysis at 60 h post-infection. To compare the incorporation of RVG and RVGTM into NDV virions, allantoic fluid was harvested from specific pathogen-free (SPF) chicken eggs at 72 h post-infection, and virus particles were purified as described previously [19 (link)]. We determined the amounts of cell-associated protein and purified virion protein by assessing the thickness of β-actin and NDV bands on western blots. Equal amounts of protein were analyzed by western blotting with SDS-12% PAGE. After being incubated with chicken serum against NDV or rabbit serum against RV, horseradish peroxidase-conjugated rabbit anti-chicken IgG (A9046; Sigma) or goat anti-rabbit IgG (ZB-2301; ZSGB-BIO) was used to detect chicken or rabbit serum binding.
5
Indirect Immunocytochemistry for PaBV-4 N Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Indirect immunocytochemistry assay, to detect PaBV-4 nucleoprotein (N protein), was conducted similar to a previously described method [4 (link)]. Briefly, the DEF cells were washed twice for 5 min each time in 0.02 M PBS, fixed for 10 min in 2% paraformaldehyde in 0.02 M PBS, and then washed twice for 5 min each time in 0.02 M PBS. Cells were permeabilized using 1% Triton X-100/0.02 M PBS for 10 min and then washed 3 times for 5 min each time in 0.03% Tween/0.02 M PBS. Blocking was performed for 2 hours in 5% dried milk/0.03% Tween/0.02 M PBS. The primary antibody, chicken IgG anti-PaBV-4 N protein, at a 1:500 dilution in 1% dried milk/0.03% Tween/0.02 M PBS, was added to the cells and then incubated in a humidified chamber for 30 min at 37°C.
Cells were washed 3 times for 5 min each time in 0.03% Tween/0.02 M PBS. Secondary antibody, horseradish peroxidase conjugated rabbit anti-chicken IgG (Sigma-Aldrich) at a 1:500 dilution in 1% dried milk/0.03% Tween/0.02M PBS, was added to the cells and incubated in a humidified chamber for 30 min at 37°C. Cells were washed 3 times for 5 min each time in 0.03% Tween/0.02M PBS and then rinsed in distilled water.
Cells were washed 3 times for 5 min each time in 0.03% Tween/0.02 M PBS. Secondary antibody, horseradish peroxidase conjugated rabbit anti-chicken IgG (Sigma-Aldrich) at a 1:500 dilution in 1% dried milk/0.03% Tween/0.02M PBS, was added to the cells and incubated in a humidified chamber for 30 min at 37°C. Cells were washed 3 times for 5 min each time in 0.03% Tween/0.02M PBS and then rinsed in distilled water.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!