Streptavidin horseradish peroxidase

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.

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.

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.