C-Peptide ELISA Kits

C-peptide is produced simultaneously to insulin. They are included in the insulin precursor - proinsulin. In proinsulin, C-peptide is located as if connecting insulin A and B chains. So, it is called "c(onnecting) peptide".

This figure shows the structure of rat proinsulin. The amino acid sequence of insulin is common to rat and mouse, however, however, a little difference in their C-peptides.
In mouse, the amino acid sequences of C-peptides corresponding to insulin I and II are as follows.

evedpqveql elggspgdlq tlalevarq (mouse I, MW 3,122)
evedpqvaql elgggpgagd lqtlalevaq q (mouse II, MW 3,134)

Usefulness of C-peptide measurement

Secretion and blood levels of C-peptide
C-peptide, after being separated from insulin, is also incorporated into secretory granules and released with insulin. It remains in the blood circulation for considerable longer than insulin though its physiological role is not clear.
So, blood C-peptide assay is used to observe biosynthetic and secretory function for insulin. It is also possible to measure C-peptide in the urine as C-peptide is excreted in urine at high concentration that is closely related to the blood level.
By measuring C-peptide, we can estimate insulin secretion in cases when insulin antibody is present in blood.

Estimation of insulin secretion by cultured cells in the presence of exogenous insulin

Measurement of C-peptide is also useful for the estimation of insulin secretion from cultured islet cells, because very often insulin is added to the culture medium exogenously. In such culture system, it is impossible to measure secreted insulin apart from that added to the culture medium. As C-peptide is produced and secreted in equimolar base to insulin, we can estimate the secreted insulin by measuring C-peptide. Deduction of exogenous insulin from the total insulin would be another way to estimate insulin secretion. But it makes big variation and also influence by the ratio of secreted insulin to exogenous insulin. So, the value would not be trustable.
Our C-peptide assay kit recognize the common amino acid sequence to C-peptide I and II, we can estimate total the amount of C-peptides.

Is there any influence of co-existing proinsulin on C-peptide measurement?

Proinsulin is processed by an enzyme at the position where two basic amino acids (K, R) moieties are present side by side as shown by black arrows in the above figure, yielding equimolar insulin and C-peptide. Most proinsulin was processed while it passes Golgi apparatus to secretory granules, however, some remains without processing and present in secretory granules, and secreted into the blood circulation with insulin and C-peptide.
When we measure insulin or C-peptide in the blood, we have to consider that we also measure the amount proinsulin because the structure of proinsulin includes those of insulin and C-peptide, if we ignore the three-dimensional structure.
We examined this possibility, and got the following results.

• Cross reactivity of rat C-peptide ELISA kit (U-type) to C-peptides, insulin, and proinsulin of various animals

  Species	Substance	Cross reactivity(%)*
  Rat	C-peptide	100
  	Insulin	Less than sensitivity
  	Pro-Insulin	Less than sensitivity
  Mouse	C-peptide	75
  	Insulin	Less than sensitivity
  Human	C-peptide	85
  	Insulin	Less than sensitivity
  	Pro-Insulin	Less than sensitivity
  Pig	Insulin	Less than sensitivity
  Cow	Insulin	Less than sensitivity

  *Cross-reactivity was estimated with 3,000 pg/ml of the substance.

• Cross reactivity of mouse C-peptide ELISA kit (U-type) to C-peptides, insulin, and proinsulin of various animals

  Animals	Substances	Crossreactivity (%)
  Mouse	C-peptide I	100
  	C-peptide II	100
  	Insulin	Less than sensitivity
  	Pro-Insulin	Less than sensitivity
  Rat	C-peptide	90
  	Insulin	Less than sensitivity
  	Pro-Insulin	Less than sensitivity
  Human	C-peptide	95
  	Insulin	Less than sensitivity
  	Pro-Insulin	Less than sensitivity

  *Cross reactivity was estimated at 3,000pg/ml of the substances.

These results clearly show that C-peptide ELISA kits can measure only C-peptides in the presence of proinsulin or insulin in assay samples.

• Measurement range 46.9～3000 pg/mL
• Measurable in a short period (5 hours)
• A minute quantity of sample is measurable (10 μL with a standard method of operating)
• All reagents are solution types and are ready to use
• High measurement accuracy and reproducibility

1) Antibody-coated plate (8 x 12) wells/plate	1 plate
2) Standard C-peptide solution (6,000 pg/mL)	500 μL
3) Buffer solution	60 mL
4) Biotinylated anti C-peptide Antibody	100 μL
5) HRP-conjugated streptavidin	100 μL
6) TMB Solution	12 mL
7) Stop Solution	12 mL
8) Wash Solution (x 10)	100 mL
9) Plate Seal	4 seals

• Cross-reactivity of mouse C-peptide ELISA kit

  Sample	Substance	Cross reaction (%)
  Mouse	C-peptide1	100
  	C-peptide2	100
  	Insulin	Less than sensitivity
  	Proinsulin	Less than sensitivity
  Rat	C-peptide	89
  	Insulin	Less than sensitivity
  	Proinsulin	Less than sensitivity
  Human	C-peptide	85
  	Insulin	Less than sensitivity
  	Proinsulin	Less than sensitivity
  Porcine	Insulin	Less than sensitivity
  Bovine	Insulin	Less than sensitivity

• Cross-reactivity of rat C-peptide ELISA kit

  Sample	Substance	Cross reaction (%)
  Rat	C-peptide	100
  	Insulin	Less than sensitivity
  	Proinsulin	Less than sensitivity
  Mouse	C-peptide	92
  	Insulin	Less than sensitivity
  	Proinsulin	Less than sensitivity
  Human	C-peptide	90
  	Insulin	Less than sensitivity
  	Proinsulin	Less than sensitivity
  Porcine	Insulin	Less than sensitivity
  Bovine	Insulin	Less than sensitivtiy

* Guideline of washing volume: 300 μL/well
**Guideline of shaking: 600-1200 rpm for 10 seconds × 3 times