1.        Bring all reagents and samples to room temperature (20-25°C) and mix gently before beginning the test.
2.        Have all reagents and samples ready before the start of the assay. Once the test has begun, it must be performed without any interruption to get the most reliable and consistent results.
3.        Use new disposable tips for each specimen.


1.        Secure the desired number of coated wells in the holder. Mark data sheet with sample identification.
2.        Dispense 25 ul of serum sample, controls and reference into the assigned wells.
3.        Dispense 100 ul of Enzyme conjugate into each well and mix for 5 seconds.
4.        Incubate for 30 minutes at 25°C
5.        Remove incubation mixture and rinse the wells five times with tap water.
6.        Dispense 100 ul of Solution A and then 100 ul of Solution B into each well.
7.        Incubate for 15 minutes at R.T.
8.        Stop reaction by adding 50 ul of 1N sulfuric acid to each well and read O.D. at 450 nm with a microwell reader.


Any microwell reader capable of determining absorbance at 450 nm may be used. The C-Peptide value of patient is obtained as follows:
1.        Plot the concentration (X) of each Reference Standards against its absorbance (Y) on graph paper.
2.        Obtain the C- PEPTIDE values of samples by reference to the Standard curve.

Well No. Description (ng/mL) Absorbance (450 nm) C-Peptide (ng/mL)
A1 0 0.005
B1 0.5 0.13
C1 2.5 0.627
D1 5 1.095
E1 10 2.033
F1 20 2.834
G1 Patient A 0.279 1.1
H1 Patient B 2.431 13.5

1.        Ashby, J.P. and B.M. Frier, Circulating C-Peptide: Measurement and Clinical Application, Annals of Clinical Biochemistry, 18:125 (1981)
2.        Yue, D.K., R.C. Baxter and J.R. Turtle, C-Peptide Secretion and Insulin Antibodies as Determinants of Stability in Diabetes Mellitus, Metabolisn, 27(1): 1978
3.        Krause, U.B., Von Erdmann, W. Atzopodien and J. Beyer, C-Peptide Measurement: A Simple Method for the Improvement of Specificity, Journal of Immunology, 2:33 (1981)