C-peptide is a 31 amino acid peptide which is a part of proinsulin and is cleaved out proteolytically during the formation of insulin. The half-life of C-peptide is longer (20-30 min) than that of insulin (3-5 min), so C-peptide is a more stable parameter while assessing b-cell function1.

This assay is a quantitative sandwich ELISA. The immunoplate is pre-coated with a monoclonal antibody specific for human C-peptide. Standards and samples are pipetted into the wells and any human C-peptide present is bound by the immobilized antibody. After washing away any unbound substances, a biotin labelled monoclonal antibody specific for human C-peptide is added to the wells. After wash step to remove any unbound reagents, streptavidin-HRP conjugate (STP-HRP) is added. After the last wash step, an HRP substrate solution is added and colour develops in proportion to the amount of human C-peptide bound initially. The assay is stopped and the optical density of the wells determined using a microplate reader. Since the increases in absorbance are directly proportional to the amount of captured human C-peptide, 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 set of sample assay. 

 

Human C-peptide (pg/mL)	Absorbance (450 nm)	Blanked Absorbance
0	0.063	0
15.6	0.117	0.054
31.2	0.182	0.119
62.5	0.298	0.235
125	0.52	0.457
250	0.934	0.871
500	1.576	1.513
1000	2.533	2.47

 

B. Sensitivity

The lowest level of C-peptide hat can be measured by this assay is 15.6  pg/mL.

 

C. Precision

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

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