Insulin is a peptide hormone exclusively produced in pancreatic beta-cells. It consists of A chain and B chain, which are linked by two disulfide bridges.
Insulin is the primary hormone responsible for glucose metabolism. Impaired insulin secretion and insulin resistance are key causes of type 2 diabetes (T2D).
PRINCIPLE OF THE ASSAY
This assay is a two-site ELISA. The micro-plate is pre-coated with a monoclonal antibody against insulin. Standards and samples are added into the wells and coincubated with a monoclonal antibody conjugated to horseradish peroxidase (HRP) enzyme. After wash step to remove any unbound substances, TMB substrate is added and colour develops in proportion to the amount of insulin bound initially. The assay is stopped and the optical density of the wells determined using a micro-plate reader. Since the increases in absorbance are directly proportional to the amount of captured insulin, the unknown sample concentration can be interpolated from a reference curve included in each assay.
TYPICAL STANDARD CURVE
The following standard curve is provided for demonstration only. A standard curve should be generated for each assay.
Insulin standard curve (log-log)
The lowest insulin level that can be measured by this assay is 0.5 ng/ml.
Intra-assay Precision (Precision within an assay) C.V. < 10%.
Inter-assay Precision (Precision between assays) C.V. <10%.
The recovery of the assay was determined by adding various amounts insulin to a sample. The measured concentration of the spiked sample in the assay was compared to the expected concentration. The average recovery was 91%.