The GM1 binding assay was performed as described previously (Li et al., 2022 (link)). Briefly, a 96-well plate was coated with 100 µL of GM1 solution (Sigma; 2 μg/mL) overnight at 4°C. After washing with PBST (PBS containing 0.05% Tween), 200 µL of blocking solution (5% skim milk in PBST) was added to each well. After incubating at 37°C for 2 h, the plate was washed again. Then, 100 µL of samples at different dilutions were added and incubated at 37°C for 1 h. After washing again, 100 µL of anti-CTB antibody was added to each well and incubated at 37°C for 1 h. After another washing step, 100 µL of HRP-labeled goat anti-rabbit antibody (1:5,000) was added and incubated at 37°C for 1 h. The plate was washed again and the soluble TMB Kit (CWbio, Beijing, China) was used for color development. The absorbance at a wavelength of 450 nm was measured using a microplate spectrophotometer.
Soluble tmb kit
Manufactured by CWBIO
Sourced in China
The Soluble TMB Kit is a laboratory reagent used in colorimetric assays. The kit provides a soluble 3,3',5,5'-Tetramethylbenzidine (TMB) substrate solution, which can be used to detect and quantify the presence of various analytes, such as enzymes or proteins, in a sample.
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6 protocols using soluble tmb kit
1
GM1 Binding Assay for Cholera Toxin
2
Quantification of Brucella Antibody Titers
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First, the 96-well plates were coated with 100 µL of B. abortus A19 LPS (100 μg/mL) and incubated overnight at 4°C. Then, the plates were washed with PBST 3 times, and 200 μL of blocking buffer (5% skim milk in PBST) was added to each well and incubated at 37°C for 2 h. After blocking, sera from each mouse were added to the corresponding wells and serially diluted with dilution buffer (10% blocking buffer) and incubated at 37°C for 1 h. The plates were washed and 100 μL of HRP-conjugated goat anti-mouse IgG, IgG1, IgG2a, IgG2b, or IgG3 antibody (Abcam, Cambridge, MA, United States) (1:15,000) was added to each well and incubated at 37°C for 1 h. After washing, the Soluble TMB Kit (CWBio, Beijing, China) was used for color development, and the absorption at a wavelength of 450 nm was measured using a microplate spectrophotometer.
3
LPS-specific Antibody Quantification
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96‐well plates were coated with LPSKpO2 extracted from K. pneumoniae stain 355 (10 µg well−1), incubated them at 4 °C overnight, washed them three times with wash buffer (PBS with 0.05% Tween 20) and dried them. Plates were blocked with blocking buffer (wash buffer with 5% skim milk powder; 200 µL well−1) at 37 °C for 2 h. After drying the plates, they were incubated in serially diluted serum (100 µL well−1) from each immunized mouse at 37 °C for 1 h. Next, plates were washed another three times and dried. Diluted HRP‐linked goat‐anti‐mouse antibodies (IgG, IgG1, IgG2a, IgG2b, and IgG3 [Abcam, Cambridge, UK]) (100 µL well−1) were added, and plates were incubated for another 1 h at 37 °C. After the washing step, a Soluble TMB Kit (CWBio, Beijing, China) was used to initiate a color‐producing reaction and measured the absorbance of each well at an OD of 450 nm.
4
Detailed ELISA Assay Protocol for LPS Detection
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A 96-well immunoplate was precoated with 10 μg/mL poly-L-lysine (100 μL per well). ELISA was performed as described previously [35 (link)]. Briefly, the 96-well immunoplate was coated for 2 h with diluted LPS at 37 °C and then washed three times with Wash Buffer (PBS + 0.05% Tween 20). The plates were patted dry and Blocking Buffer (PBS + 5% milk powder) was added to each well followed by incubation at 37 °C for 2 h. After drying the plates, diluted serum from the immunized mice was added to each well and the plate was incubated at 37 °C for 1 h. After another washing and drying step, 1:15,000 diluted HRP-conjugated goat anti-mouse IgG antibody (Abcam, Shanghai, China) was added to each well and incubated at 37 °C for 1 h. The washing and drying step was repeated. The Soluble TMB Kit (CWbio, Beijing, China) was used to initiate the detection reaction. Stop solution (2 M H2SO4) was added to each well to stop the reaction, and the absorbance of each well was measured at a wavelength of 490 nm with a microplate reader.
5
ELISA Assay for S. flexneri Antibodies
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We coated 96-well plates with LPS from S. flexneri 2a 301 (10 μg/well) overnight at 4 °C. The next day, each well was washed three times with PBST (PBS with 0.05% Tween 20) using an automated plate washer (ELx50 Washer, BioTek Instruments, Inc., Winooski, VT, USA). After washing and drying, the wells were loaded with 200 μL of blocking buffer (5% skim milk powder in PBST) and incubated at 37 °C for 2 h. Next, the wells were incubated with diluted serum at 37 °C for 1 h. After another washing and drying step, 100 μL of HRP-conjugated donkey anti-mouse IgG (Abcam, China) was added to each well, and the plates were incubated at 37 °C for 1 h. Plates were again washed and dried, and then the Soluble TMB kit (CWBio, Beijing, China) was used for color development and the reactions were stopped with 50 μL of stop solution per well. The absorbance at a wavelength of 450 nm was then measured.
6
ELISA to Detect Mouse Antibody Titers
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Enzyme-linked immunosorbent assay (ELISA) was performed to determine the antibody titres of the mouse serum. Briefly, 96-well immunoplates were coated with LPS at a concentration of 5 μg polysaccharide/well (100 μL/well) and incubated at 37°C for 2 h. Then, the wells were washed three times with PBST (PBS +0.05% Tween 20) and patted dry. Next, 200 μL of blocking buffer (PBST +5% skimmed milk powder) was added to each well and incubated overnight at 4°C. The mouse serum samples were diluted with dilution buffer (PBST +0.5% skimmed milk powder) and added to each well (100 μL/well) of the immunoplate after it was patted dry, and then the plate was incubated at 37°C for 1 h. After washing and drying, HRP-conjugated goat anti-mouse IgG, IgG1, or IgG2a (Abcam, Shanghai, China) antibody was diluted 50,000 times with dilution buffer and added to each well (100 μL/well). After another incubation, washing, and drying step, a Soluble TMB kit (CWbio, Beijing, China) was used to initiate the colour reaction. The microplate reader was used to measure the absorbance with a detection wavelength of 450 nm.
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