96-well EIA/RIA plates (Corning) were coated overnight at 4 °C with 20 μg of G protein per plate in coating buffer (Solarbio). All wells were blocked with 100 µl of blocking buffer (5% skim milk in PBS) for 1.5 h at 37 °C. After standard washes and blocks, serum (2x serially diluted, starting at 100x dilution) in 5% skim milk in PBS was incubated at 37 °C for 2 h. Plates were washed four times in PBS with 0.1% Tween 20 (PBST) before addition of HRP-conjugated goat anti-mouse IgG (Abcam, Cat No. ab6789, diluted 1:20,000), IgA (Abcam, Cat No. ab97235, diluted 1:10,000) or HRP-conjugated goat anti- hamster IgG (Abcam, Cat No. ab6892, diluted 1:15,000) diluted in 5% milk in PBS for 1.5 h at 37 °C. Plates were washed as before prior to being developed with 100 µl/well of TMB chromogen solution (Beyotime) for 15 min. Substrate reactions were stopped by the addition of 50 µl/well of stop solution for TMB Substrate (Beyotime) before reading plate absorbance at 450 nm (OD450). The cutoff value was defined as 2.1-fold of OD450 values from the sample of nonvaccinated mice. The reciprocal of the maximum sample dilution with OD450 values equal to or greater than the cutoff value was used to calculate the endpoint binding antibody titers.
Coating buffer
Manufactured by Solarbio
Sourced in China
Coating buffer is a solution used to prepare the surface of a solid substrate for subsequent coating or immobilization of biomolecules, such as proteins or antibodies. It provides a suitable environment for the adsorption or covalent attachment of these molecules to the substrate.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti mouse igg, by Abcam (1 mentions)
Tmb substrate, by Beyotime (1 mentions)
Eia ria plate, by Corning (1 mentions)
Hrp conjugated goat anti hamster igg, by Abcam (1 mentions)
Tmb chromogen solution, by Beyotime (1 mentions)
Igg2c, by Southern Biotech (1 mentions)
Hrp conjugated anti mouse igg, by Southern Biotech (1 mentions)
Elisa kit, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
Stop buffer, by Solarbio (1 mentions)
Tmb solution, by Solarbio (1 mentions)
Phosphate buffered saline (pbs), by Solarbio (1 mentions)
6 protocols using coating buffer
1
ELISA Protocol for Antibody Titer Determination
2
Epitope Mapping of Immunodominant Peptide
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To identify the core epitopes of the immunodominant epitope peptide, we designed different truncated peptides that could cover its full length (Figure 2 ). These truncated peptides were synthesized by Jill Biochemical (Shanghai, China). ELISA plate was coated with these five truncated peptides (50, 100, 200, 400 ng/well, respectively), irrelevant negative control peptide (GST141−158), positive control peptide Sao355−384, negative control protein (BSA), positive control rSao-M protein, and in coating buffer (Solarbio) overnight at 4°C. Then wells were blocked with 3.0% BSA in PBS. After the wells were washed, anti-immunodominant peptide serum of four different dilutions (1:1000,1:2000,1:4000,1:8000, diluted with PBS containing 1% BSA) were incubated in the wells for 1 h at 37°C. The next ELISA steps were the same as described above.
3
Virus-Specific Antibody Detection in Mice
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For the detection of virus-specific antibodies in mouse serum, 96-well ELISA plates were coated overnight with 100 μl per well of 2 μg/ml M2e peptides (SLLTEVETPIRNEWGSRSNDSSD) or HA2 peptides (NAELLVL) in coating buffer (Solarbio, Beijing, China). After 3 washes in PBST (PBS with 0.1% Tween 20), plates were blocked using 300 μl of 3% BSA (bovine serum albumin) for 1 h at 37°C. The diluted serums were added to the plate for 1 h at 37°C. Plates were washed three times and incubated with HRP-conjugated anti-mouse IgG (1:5,000), IgG1 (1:5,000), and IgG2c (1:5,000) (SouthernBiotech, Birmingham, AL, United States). After washing three times, the TMB substrate was added to each well and incubated for 10 min. Then, the reaction was stopped by adding a stop solution. The optical density (OD) was read at 450 nm by a microplate reader (Molecular Devices, San Jose, CA, USA). Cytokines in serum, such as IL-4, IL-2, and IFN-γ, were detected by using the corresponding ELISA kits (Thermo Fisher Scientific, Waltham, MA, United States).
4
ELISA Detection of S. suis 2 Antibodies
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The ELISA method was used for preliminary analyses of whether the core epitope had the ability to detect S. suis 2 infection. ELISA plate was coated with the core epitope (100 ng/well) in coating buffer (Solarbio) overnight at 4°C. After the wells were blocked with 3.0% BSA and washed, human serum from different sources of four dilutions (1: 200, 1: 400, 1: 800, 1: 1600, diluted with PBS containing 1% BSA) were added and the ELISA plate was incubated at 37°C for 20 min. After all unbound material was washed off, a peroxidase-conjugated goat anti-human IgG (H+L; Biodragon) was added for 1 h. The next ELISA steps were the same as described above.
5
Serum Antibody Detection of FMDV
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The detection of serum antibodies against FMDV was performed by enzyme-linked immunosorbent assay (ELISA). The inactivated FMDV (purified 146 S particles) was coated with coating buffer (Solarbio Life Science, Beijing, China) and incubated overnight at 4 °C. After being blocked with 5% skim milk for 1 h at 37 °C, the plate was washed three times with PBS-0.1% Tween 20 (PBST) and incubated with serial twofold dilutions of each serum sample prepared in PBS, for 1 h at 37 °C. After six washes with PBST, the plates were incubated at 37 °C for 45 min with HRP-conjugated goat anti-mouse IgG (ImmunoWay, USA) for mouse sera, and HRP-conjugated rabbit anti-guinea pig IgG (ImmunoWay, USA) for guinea pig sera, both at a 1:5000 dilution in PBST. After another wash, 100 µL of 3, 39, 5, 59-tetramethylbenzidine was added to each well as a chromogenic substrate solution. The reaction was stopped with 50 µL of 0.5 M H 2 SO 4 after incubation at room temperature for 30 min. The optical density at 450 nm (OD value) of each well was measured using an ELISA reader.
6
SARS-CoV-2 Antibody Response Monitoring
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Serum samples were collected at 0, 7, 14, 21, 28, and 35 dpv. Total IgG was detected by enzyme-linked immunosorbent assay (ELISA). The inactivated viruses were coated with coating buffer (Solarbio Life Science, Beijing, China) and incubated overnight at 4℃. The coated wells were blocked with 5% skim milk and incubated at 37℃ for 1 h, discarding the skim milk, TBST was used to wash the coated wells twice. The serum samples (experimental groups and negative controls) were diluted with PBS (Solarbio Life Science, Beijing, China), added into the wells of ELISA plates, and incubated at 37℃ for 1 h. The serum samples were removed from the coated wells, and then TBST was used to wash the wells (three times, shaking for 3 min). HRP goat anti-mouse antibody (Beyotime Biotechnology, Shanghai, China) was used as the secondary antibody (1:10000) and incubated at 37℃ for 30 min. The wells were washed with TBST five times (shaking for 3 min). TMB solution (Solarbio Life Science, Beijing, China) was added into the wells, incubated at room temperature for 15 min (keep in the dark), and then the reaction was stopped by stop buffer (Solarbio life science, Beijing, China). The absorbance was read at 450 nm (OD value). The titers of the antibody were calculated when S/P ≥ 2.1 (S/P = OD450 nm of experimental groups / OD450 nm of negative controls).
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