The SARS-CoV PUMC01 strain 46 (link) was diluted with coating buffer to 4 × 10−5 TCID50/mL (bicarbonate/carbonate coating buffer, 0.05 M, pH 9.6). The virus was then coated on 96-well plates with 100 μL (4 × 10−4 TCID50) of virus per well for 12 h at 4 °C. The coating buffer was removed from the wells and 100 μL of blocking buffer was added per well and incubated for 12 h at 4 °C (2% BSA in PBS, pH 7.4. GIBCO, Carlsbad, USA). The plates were washed twice with PBS (pH 7.2). The mouse monoclonal antibodies against SARS-CoV and control mAb against HIV-P27 were diluted to 0.001 μg/μL in the blocking buffer, added to the wells separately and incubated for 1 h at 37 °C. The plates were washed twice with PBS and the secondary antibody (HRP-conjugated goat anti-mouse IgG, (GeneTex, Irvine, USA) diluted 1:1000 in blocking buffer) was added to each well and incubated for 1 h at 37 °C. The secondary antibody was removed and the plate was washed three times with PBS, then 100 μL of TMB substrate solution (Promega, Madison, WI, USA) was added to each well. After incubation for 30 min at room temperature, the absorbance of the test wells was read at 450 nm (A450).
Tmb substrate solution
Manufactured by Promega
Sourced in United States
TMB substrate solution is a colorimetric substrate used in enzyme-linked immunosorbent assays (ELISA) to detect and quantify the presence of specific analytes. It produces a blue color upon reaction with the enzyme, which can be measured spectrophotometrically.
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Lab products found in correlation
3 protocols using tmb substrate solution
1
SARS-CoV Antibody Binding Assay
2
Quantitative ELISA for SARS-CoV-2 RBD and bFGF Expression in Microalgae
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Severe acute respiratory syndrome coronavirus 2 RBD antigen (250 μg/ml) purified from recombinant N. benthamiana was used as the positive control. For quantitative ELISA, crude protein extracts from C. reinhardtii and C. vulgaris were diluted in 0.2 M carbonate buffer (pH 9.6). The diluted sample was added to an ELISA plate for protein adsorption overnight at 4°C. After blocking with 5% fat-free dry milk solution for 2 h at room temperature, the plates were incubated with anti-RBD serum (1:3,000) collected from sheep immunized with synthetic partial RBD peptide (CLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQ) overnight at 4°C. The plates were subsequently incubated with horseradish peroxidase-conjugated anti-sheep IgG at a dilution of 1:5,000 (A3415, Sigma-Aldrich) for 2 h at room temperature. A colorimetric reaction was induced by the addition of the TMB substrate solution (Promega, United States) with 30 min of incubation at 25°C. The absorbance values were measured at 450 nm. Standard curves were constructed using pure RBD protein from N. benthamiana to estimate the expression levels.
The bFGF expressed in transformed C. vulgaris and C. reinhardtii was quantified using the human FGF basic ELISA Kit (R&D Systems). The protocol was followed according to the manufacturer’s instructions.
The bFGF expressed in transformed C. vulgaris and C. reinhardtii was quantified using the human FGF basic ELISA Kit (R&D Systems). The protocol was followed according to the manufacturer’s instructions.
3
SARS-CoV-2 RBD Antibody ELISA
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SARS-CoV-2 RBD antigen (Sino Biological, Inc., Beijing, China) were used to coat microtiter plates at a concentration of 500 ng/mL in PBS (0.01 M, pH 7.4) and incubated at 4°C overnight. Plates were washed and blocked using 250 μL of blocking buffer (5% skim milk in PBS-Tween) at room temperature for 1 h followed by washing three times with wash buffer. Purified IgY antibody samples from immunized and non-immunized hens were serially diluted starting from a 1:50 ratio in blocking buffer. Plates were then incubated at 37°C for 1 h and washed three times with PBS-Tween. Horseradish peroxidase (HRP)-conjugated rabbit anti-chicken IgY (Abcam, Cambridge, UK) at a 1:10,000 dilution was added in a 100 μL/well and incubated for 1 h at 37°C. Plates were washed and color was developed by adding 100 μL/well of TMB substrate solution (Promega, Madison, WI, USA) and incubating for 30 min. color development was stopped by adding 2M H2SO4 (100 μL/well).
Optical density (OD) was measured at a wavelength of 450 nm using ELISA plate reader (ELX800 Biokit) with PBS as a blank control and IgY extracted from non-immunized hens used as negative control. The IgY titer was defined as the maximum sample dilution that showed an OD value 2.1 times higher than that of the negative control.
Optical density (OD) was measured at a wavelength of 450 nm using ELISA plate reader (ELX800 Biokit) with PBS as a blank control and IgY extracted from non-immunized hens used as negative control. The IgY titer was defined as the maximum sample dilution that showed an OD value 2.1 times higher than that of the negative control.
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