Serum levels of IgG against Sjögren’s syndrome-related antigen A (SSA) and M3 muscarinic receptor (M3R) were examined by a standard sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well MaxiSorp plates were coated with antigen peptides (5 μg/ml) at 4 °C overnight. Plates were washed and incubated with blocking buffer (0.5% gelatin, 0.5% bovine serum albumin and 0.05% Tween 20 in PBS) at room temperature for 1 h. Serum samples were diluted (1:100) and incubated in plates for 2 h at room temperature, followed by incubation of biotin-conjugated anti-mouse IgG (BioLegend, 0.5 μg/ml) for 1 h. After washing, HRP Streptavidin (BioLegend, 1:1000) was added, and the plates were incubated for 30 min. Then, plates were washed and freshly prepared TMB substrate (BioLegend, 50 μl) was added. After 10 min, 20 μl stop solution (3 M H2SO4) was added and absorbance at 450 nm was measured using a Sunrise microplate reader (Tecan, Männedorf, Switzerland). Antigenic peptides of SSA (AVALREYRKKMDIPA) and M3R (VLVNTFCDSCIPKTYWNLGY).
一般情况下， Antigenic多肽s of M3R 粉末应该保存在-20甚至-70摄氏度. Antigenic多肽s of M3R 溶解后应该-70摄氏度分装保存，避免反复冻融. 了解更多细节，请查阅手册:多肽溶解及保存指南
Xiao F, Lin X, Tian J, Wang X, Chen Q, Rui K, Ma J, Wang S, Wang Q, Wang X, Liu D, Sun L, Lu L. Proteasome inhibition suppresses Th17 cell generation and ameliorates autoimmune development in experimental Sjögren's syndrome. Cell Mol Immunol. 2017 Jul 10;14(11):924–34. doi: 10.1038/cmi.2017.8. Epub ahead of print. PMID: 28690324; PMCID: PMC5675963.