Microtitre plates (Coaster, Cambridge, MA, USA) were coated with R97-116 peptide (5 μg/ml) at 4 °C over night. Then, the plates were blocked with 200 μl of PBS containing 0.05 % Tween 20 and 10 % FBS at 37 °C for 1.5 h. Serum samples (1:100 in PBS/0.05 % Tween 20) were added to the wells and incubated for 2 h at 37 °C. After washing, the plates were incubated for 1 h with biotinylated rabbit anti-rat IgG (1:3000; Biosynthesis Biotechnology, Beijing, China). Then, streptavidin-horseradish peroxidase (1:1000; Biosynthesis Biotechnology) was added, incubated at 37 °C for 30 min. Plates were washed with PBS containing 0.05 % Tween 20 and followed by development with tetramethylbenzidine (TMB) substrate (Tiangen Biotechnology, Beijing, China). Finally, OD value into each well was measured at 450 nm subtracted from 630 nm using a microplate ELISA reader. Each serum was tested in triplicate. Results were expressed as mean OD value of samples ± SD.
Tetramethylbenzidine tmb substrate
Manufactured by Tiangen Biotech
Sourced in China
Tetramethylbenzidine (TMB) substrate is a widely used colorimetric substrate for the detection and quantification of peroxidase enzyme activity. It is commonly used in various immunoassay techniques, such as enzyme-linked immunosorbent assays (ELISA), to visualize the presence and concentration of target analytes.
Automatically generated - may contain errors
4 protocols using tetramethylbenzidine tmb substrate
1
ELISA for Rat IgG Detection
2
Rat Anti-AChR97–116 Antibody Assessment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rat serum anti-AChR97–116 IgG, IgG1, IgG2a, and IgG2b antibody levels were detected by ELISA as described previously [17 (link)]. The following reagents were used: biotin rabbit anti-rat IgG (Poly4054; BioLegend), IgG1 (MRG1-58; BioLegend), IgG2a (MRG2a-83; BioLegend), IgG2b (MRG2b-85; BioLegend), streptavidin-horseradish peroxidase (Biosynthesis Biotechnology), and tetramethylbenzidine (TMB) substrate (Tiangen Biotechnology, Beijing, China). The serum anti-AChR97–116 IgG antibody affinities were determined by the thiocyanate method as described previously [19 (link)]. OD values were read at a wavelength of 450 nm by using a microplate ELISA reader.
3
Serum-based Immunoassay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flat-bottomed polystyrene 96-well plates (Corning, USA) were coated with 100 μl BPM (10 μg/ml in PBS) overnight at 4 °C. Then, the plates were blocked with 10% FBS. Diluted serum (1: 100) was added and incubated for 2 h at 37 °C, followed by biotin-labeled anti-rat IgG (Biolegend, USA) for 1 h at 37 °C and streptavidin–horseradish peroxidase (Bios, China) for 30 min at 37 °C. The color was developed with tetramethylbenzidine (TMB) substrate (Tiangen Biotechnology, China). Finally, OD values of corresponding wells were determined by a microplate ELISA reader at 450 nm. Results were expressed as mean optical density (OD values) ± standard deviation (SD).
4
Quantification of R97-116 Peptide Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A standard ELISA technique was used to detect R97–116 peptide specific antibody. Briefly, R97–116 peptide (5 μg/ml) was coated onto a flat-bottomed 96-well microtitre plates (Corning) in 0.1 M carbonate bicarbonate buffer (pH 9.6) overnight at 4 °C. Then, the plates were blocked with 200 μl of PBS containing 0.05 % Tween20 and 10 % FCS at 37 °C for 1.5 h. A total volume of 100 μl diluted serum samples (1:100 in PBS/0.05 % Tween20) were added and incubated for 2 h at room temperature. After washed, biotin rabbit anti-rat IgG (1:3000; Biosynthesis Biotechnology, Beijing, China), biotin anti-rat IgG1, IgG2a, and IgG2b (1:500; BioLegend) were added to the wells and incubated for 1 h. Then streptavidin-horseradish peroxidase (1:1000; Biosynthesis Biotechnology) was added, incubated at 37 °C for 30 min. Then, plates were washed with PBS containing 0.05 % Tween20 and followed by development with Tetramethylbenzidine (TMB) substrate (Tiangen Biotechnology, Beijing, China). Finally, plates were read at a wave length of 450 nm using a microplate ELISA reader. Each serum was tested in triplicate. Results are expressed as mean OD value of samples ± SD.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!