The specific antibodies against P97R1 or P46 were analyzed by ELISA. Briefly, 96-well microtiter plates were respectively coated with 100 μl/well P97R1, P46 proteins (2 μg in 1 ml coating liquid) and incubated at 4°C for 12 h. The wells were then washed three times with PBST (pH 7.4, 150 mM PBS, 0.05% Tween 20) and blocked with 400 μl/well PBS (pH 7.4, 150 mM PBS) containing 5% (wt/vol) skim milk at 37°C for 2 h. The wells were washed three times with PBST, then 100 μl serum (diluted 1:200 in PBST) or 100 μl BALs (diluted 1:50 in PBST) was added to each well, and incubated at 37°C for 1.5 h. Then wells were washed three times with PBST and respectively incubated with 100 μl of 1:2000 diluted horse anti-mouse IgG (or IgG1 or IgG2a) antibodies, rabbit anti-mouse IgA antibodies (CST), which all conjugated with HRP at 37°C for 1 h. The wells were then washed three times and 100 μl of 3,3′-5,5′-tetramethyl benzidine substrate (Solarbio) was added to each well. After 7–15 min of incubation, the reaction was stopped by adding 50 μl of 2 mol/l H2SO4, and the plates were read at 450 nm with a spectrophotometer plate reader (TECAN).
3 3 5 5 tetramethylbenzidine substrate
Manufactured by Solarbio
Sourced in China
3,3′,5,5′-tetramethylbenzidine substrate is a chromogenic substrate used in enzymatic assays. It undergoes a color change reaction when catalyzed by certain enzymes, allowing for colorimetric detection and quantification of enzyme activity.
Automatically generated - may contain errors
4 protocols using 3 3 5 5 tetramethylbenzidine substrate
1
Quantifying Antibody Responses Against P97R1 and P46
2
Cytokine Profiling of T Cells
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The levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-2, IL-10 and transforming growth factor (TGF)-β secreted by T cells were detected using ELISA Max kits (SEKH-0047; SEKH-0046; SEKH-0018; SEKH-0316; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. In brief, cytokine capture antibodies were coated in 96-well plates at 4°C overnight, and the wells were then blocked at room temperature for 1 h. Cell supernatant (100 μl) was added into each well and then incubated for 2 h at 37°C. Next, 100 μl of detection antibody and diluted streptavidin peroxidase were added in succession. Finally, the reaction was stopped by adding 3,3′,5,5′-tetramethylbenzidine substrate (Beijing Solarbio Science & Technology Co., Ltd.) and sulfuric acid (Sigma-Aldrich; Merck KGaA). The optical density value at 450 nm was measured via a microplate reader (Bio-Rad Laboratories, Inc.).
3
RVFV Gn Antigen Quantification
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The RVFV purified truncated Gns (Gn1a–Gn10a) and RVFV Gn were coated at 0.2 μg per well on 96-well plates at 4 °C overnight. The plates were washed three times with PBST (0.2% Tween 20 in PBS) and blocked with 2% BSA at 37 °C for 1 h. The plates were washed three times with PBST, and the threefold serial dilutions of serum samples in PBST containing 0.2% BSA (starting at 1:100 dilution) were added for 1 h at 37 °C. Plates were washed three times with PBST, and an HRP-conjugated anti-human IgG antibody (Abcam, Cambridge, UK) was added for 1 h at 37 °C. Then, 3,3′,5,5′-tetramethylbenzidine substrate (Solarbio, Beijing, China) was added after being washed three times and 2 M H2SO4 was added to stop the reaction. The optical density (OD) was measured at 450–630 nm (OD450–OD630).
4
SARS-CoV-2 Antibody Isotype ELISA
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An enzyme‐linked immunoabsorbent assay (ELISA) was used to measure anti‐SARS‐CoV‐2 specific IgE. First, a 96‐well microtiter ELISA plate was coated with 2 μg ml−1 of recombinant SARS‐CoV‐2 S antigen and SARS‐CoV‐2 N antigen (Sino Biological) overnight at 4°C with blocking by 20% nonfat dried milk. Then, samples were added and incubated at 37°C for 2 h, and the plate was then washed three times with phosphate buffered saline containing 0.04% Tween‐20. For measurement of IgE, 50 μl of serum (not diluted in phosphate buffer saline (PBS) was incubated at 37°C for 2 h. Then, 100 μl of horse radish peroxidase (HRP) labeled goat anti‐human IgE HRP (diluted 1:5000; Abcam, ab3901) was added, and the sample was incubated at room temperature for 2 h. The plates were washed three times with PBS, and the signal was developed by adding 100 μl of the 3,3',5,5'‐ tetramethylbenzidine substrate (Solarbio) for 15 min at room temperature. The reaction was stopped by adding 50 μl of 2‐M sulfuric acid. Plates were read on a Spectramax Plate Reader at 450 nm using SoftMax Pro, and the background optical density was subtracted. The cutoff (normal) value was the average level in the healthy controls.
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