Insulin and the IGFs are members of the insulin family of molecules. IGF-I and II are structurally homologous to proinsulin, and can be thought of as taking the shape of an exaggerated, inverted letter G. Intrachain disulfide bonds link sections of the G. The single chain insulin propeptide consists of a 30 amino acid B chain (aa 2554), a C-peptide (aa 5589), and a 21 aa A chain (aa 90110). Removal of the C-peptide by proteolysis enables the formation of mature Insulin, a disulfidelinked heterodimer of the A and B chains. Circulating C-peptide levels are elevated in hyperinsulinism, obesity, and type II diabetes.

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for insulin has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any insulin present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for insulin is added to the wells and binds to the combination of capture antibody-insulin in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A coloured product is formed in proportion to the amount of insulin present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven insulin standard dilutions and insulin sample concentration determined.

Figure 1:Schematic diagram of the assay

1. Aluminium pouches with a Microwell Plate coated with antibody to human insulin (8 x 12)

2. 2 vials human insulin Standard lyophilized, 1000pmol/L upon reconstitution

3. 2 vials concentrated Biotin-Conjugate anti-human insulin antibody

4. 2 vials Streptavidin-HRP solution

5. 1 bottle Standard /sample Diluent

6. 1 bottle Biotin-Conjugate antibody Diluent

7. 1 bottle Streptavidin-HRP Diluent

8. 1 bottle Wash Buffer Concentrate 20x (PBS with 1% Tween-20)

9. 1 vial Substrate Solution

10. 1 vial Stop Solution

11. 4 pieces Adhesive Films

12. package insert

NOTE: [96 Tests]

Table 1: Storage of the kit

**Provided this is within the expiration date of the kit.

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