The detection of the sera IgG level and the SIgA level in the vaginal lavage, intestinal tract, and faecal pellets, was performed by indirect ELISA. PoRV propagated on MA104 cells were utilized as an antigen with which to coat polystyrene microtiter plates, which were then incubated overnight at 4°C. Cell cultures were used as negative controls for the antigen. After discarding the waste solution, the well plates were washed three times with PBS containing 0.05% Tween 20 (PBST) for 5 min and blocked with 5% skim milk in PBS at 37°C for 2 h. After the well plates were washed three times with PBST, the collected samples were incubated for 2 h at 37℃, and each sample repeated three times. The serum was diluted 10 times with 5% skim milk, and the vaginal lavage, intestinal tract, and faecal lysate were centrifuged to collect supernatant for use. Subsequently, an HRP-conjugated goat anti-mouse IgG or IgA antibody (Invitrogen, USA) was added to wells with a dilution of 1:5000 and incubated for 1 h at 37°C. The colour development was carried out under dark conditions with a TMB solution at a ratio of 1:1, and the OD450 value was determined.
Hrp conjugated goat anti mouse igg or iga antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The HRP-conjugated goat anti-mouse IgG or IgA antibody is a secondary antibody conjugated with horseradish peroxidase (HRP). It is designed to detect and bind to primary antibodies raised in mice, specifically immunoglobulin G (IgG) or immunoglobulin A (IgA). The HRP conjugation allows for colorimetric or chemiluminescent detection in various immunoassay techniques.
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5 protocols using hrp conjugated goat anti mouse igg or iga antibody
1
IgG and SIgA Detection in Mucosal Samples
2
TGEV and PEDV Antibody Detection
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To detect TGEV- and PEDV-specific antibodies in the collected samples, polystyrene microtiter plates were coated with purified TGEV/PEDV for 12 h at 4 °C, using cultured ST/Vero cells as a negative antigen control. After blocking with 5% skim milk at 37 °C for 2 h and washing three times with PBS-0.1% Tween 20 (PBST), serum and mucus samples serially diluted in PBS-1% BSA were added to wells in triplicate, and then plates were incubated for 1 h at 37 °C. After washing with PBST, HRP-conjugated goat anti-mouse IgG or IgA antibody (Invitrogen, Carlsbad, CA, USA) was added to each well (1:5000) and incubated for an additional 1 h at 37 °C. The substrate o-phenylenediamine dihydrochloride (Sigma) was used for color development, and the absorbance was measured at 490 nm.
3
PEDV IgG and IgA ELISA Assay
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The levels of IgG in the sera and IgA in the external genital tract, intestinal tract, and stools were measured by ELISA. Polystyrene microtiter plates were coated overnight at 4 °C with PEDV NJ propagated on Vero cells and the culture of Vero cells used as a negative control for the antigen. After blocking with 5% skimmed milk, the collected samples were serially diluted in PBS, added in triplicate and incubated at 37 °C for 1 h. Then, an HRP-conjugated goat anti-mouse IgG or IgA antibody (Invitrogen, USA) was added to each well (1:5000) and incubated for 1 h at 37 °C. Color was then developed using o-phenylenediamine dihydrochloride (Sigma, USA) as a substrate, and absorbance at OD490 was measured.
4
Measurement of IgG and IgA by ELISA
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The levels of IgG in the sera and IgA in the feces were measured by the ELISA methods with some modification [18 (link), 23 (link)]. The methods were as follows: Polystyrene microliter plates were coated overnight at 4 °C with 100 μL 10 μg/mL PEDVS protein, OMP16-PEDVS protein, or 100 μL recombinant L. casei-OMP16-PEDVS strain. After blocking with 5% skimmed milk, the collected samples were serially diluted in PBS, added in triplicate, and incubated at 37 °C for 1 h. Then, an HRP conjugated goat anti-mouse IgG or IgA antibody (Invitrogen, USA) was added to each well (1:5000) and incubated for 1 h at 37 °C. The polystyrene microtiter plates were washed 5 times during each step. At last, 100 μL of TMB substrate (tetramethylbenzidine and H2O2) was added to each well and 50 μL of stop solution was added after 10 mins. The OD values at 630 nm were measured using a multimode plate reader (EnVision).
5
SARS-CoV-2 Spike Protein Antibody Assay
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Interleukin (IL)-4, IL-10, and interferon (IFN)-γ in the serum of immunized mice were detected using commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA). Standard curves were generated for each cytokine. Values were measured at an optical density of 450 nm using an ELISA plate reader (SpectraMax M2e, Molecular Devices, San Jose, CA, USA). Antigen-specific immunoglobulin (Ig)G from serum or IgA from fecal samples was measured using ELISA based on our previous study (Hwang et al., 2023 (link)). Briefly, each well of a 96-well plate was pre-coated with E. coli-expressed recombinant SARS-CoV-2 spike S1 antigens (1 µg per well) overnight at 4°C. The antigen-coated wells were blocked with bovine serum albumin for 1 h at 37°C. After blocking, immunized mouse serum (1:100 dilution) or fecal extract (1:10 dilution) was added to the wells and incubated at 37°C for 1 h. HRP-conjugated goat anti-mouse IgG or IgA antibody (1:1000; Invitrogen, Waltham, MA, USA) was added to the wells and incubated for 1 h at 37°C. The antigen-specific antibody was detected by adding 3, 3’, 5, 5’- tetramethylbenzidine substrate (Sigma-Aldrich, St. Louis, MO, USA). The reaction was stopped by adding 0.5 N H2SO4. Optical density (450 nm) was measured using an ELISA plate reader (SpectraMax M2e, Molecular Devices, San Jose, CA, USA).
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