ELISA Quantification of RDK2 IgG

The performance of the recombinant RDK2 was evaluated by ELISA, and the levels of IgG antibodies (specific for antigen) were detected by enzyme-linked immunosorbent assay (ELISA). The experiment was performed by coating recombinant RDK2 antigen (1 mg/mL) in 96-well ELISA plates (Nunc) overnight at 4 °C diluted in phosphate buffer (PB). The next day, the wells were blocked with 1% bovine serum albumin (BSA) (200 µL/well) at 37 °C for 1 h. For primary antibody binding, serum (100 µL/well) and urine samples (100 µL/well) were added at a dilution of 1:2000 [19 (link)] and 1:10 [16 (link)], respectively, and incubated at 37 °C for 2 h, followed by secondary antibodies at a 1:3000 dilution of horseradish peroxidase (HRP)-conjugated anti-human IgG. Finally, the presence of bound IgG was detected by adding 3,3’,5,5’-Tetramethylbenzidine (TMB) (Sigma-Aldrich, St. Louis, MI, USA), as the substrate, and the reaction was stopped by the addition of 2N H₂SO₄. Optical density values were obtained at 450 nm using a microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).