Summary:The insulin enzyme-linked immunosorbent assay (ELISA) is a standard immunoassay for the detection of insulin levels in serum samples. This experiment uses a 96-well plate coated with anti-insulin antibodies and developed with a secondary anti-insulin antibody linked to horseradish peroxidase. The ELISA is developed with 3,3',5,5'-Tetramethylbenzidine (TMB), a chromogenic substrate and read on an automated plate spectrophotometer at 450 nm. Insulin ELISA may be processed with normal or ultra-sensitive assay kit depending on the expected range of sample concentrations. Serum insulin levels are affected by pancreatic \u03b2-cell function and insulin sensitivity. Serum insulin levels are elevated in obesity and insulin resistance.