Insulin is a hormone synthesized by the b cells of pancreatic islets. It consists of two amino acid chains, A chain and B chain, linked with a sulphide bond. The A chain is made of 21 amino acids and B chain with 30 amino acids. Insulin has a molecular weight of 5.8KDa.

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 co-incubated 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.

A. Typical representation of standard curve

The following standard curve is provided for demonstration only. A standard curve should be generated for each assay.

 

Insulin (µU/mL)	Absorbance (450 nm)	Blanked Absorbance
0	0.052	0
3	0.074	0.022
6	0.114	0.062
12	0.226	0.174
25	0.549	0.497
50	1.189	1.137
120	2.705	2.653

 

B. Sensitivity

The lowest insulin level that can be measured by this assay is 3 µU/mL.

 

C. Precision

Intra-assay Precision (Precision within an assay) C.V. < 10%.

Inter-assay Precision (Precision between assays) C.V. <10%.

 

D. Recovery

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 92%.