Chemiluminescence Immunoassay (CLIA) detection using Microplate luminometers provides a sensitive,
high throughput, and economical alternative to conventional colorimetric methodologies, such as Enzyme-linked immunosorbent assays (ELISA).
Diabetes is one of the leading causes of disability and death in the U.S. It affects an estimated 16 million
Americans, about one third of them do not even know they have the disease. The causes of diabetes are
not precisely known, but both genetic and environmental factors play a significant role. The disease is
marked by deficiencies in the body’s ability to produce and properly use insulin. The most common forms of diabetes are type 1, in which the body’s ability to produce insulin is destroyed, and type 2, in which the body is resistant to insulin even though some amount of insulin may be produced.
In-vitro determination of insulin and C-Peptide levels help in the differential diagnosis of liver disease,
acromegaly, Cushing’s syndrome, familial glucose intolerance, insulinoma, renal failure, ingestion of
accidental oral hypoglycemic drugs or insulin induced factitious hypoglycemia. Both insulin and CPeptide
are produced by enzymatic cleavage of proinsulin. Proinsulin is stored in the secretory granules
of pancreatic β-cells and is split into a 31 amino acid connecting peptide (C-Peptide; MW 3600) and
insulin (MW 6000). C-Peptide is devoid of any biological activity but appears to be necessary to maintain
the structural integrity of insulin. Although insulin and C-Peptide are secreted into portal circulation in
equimolar concentrations, fasting levels of C-Peptide are 5-10 fold higher than those of insulin owing to
the longer half-life of C-Peptide. The liver does not extract C-Peptide however; it is removed from the
circulation by degradation in the kidneys with a fraction passing out unchanged in urine. Hence urine CPeptide levels correlate well with fasting C-Peptide levels in serum. The glucagon stimulated C-Peptide
determination is often used for differential diagnosis of insulin-dependent from non-insulin-dependent
The C-Peptide Quantitative Test Kit is based on a solid phase enzyme-linked immunosorbent assay.
The assay system utilizes one anti-C- Peptide antibody for solid phase (microtiter wells) immobilization
and another anti-C-Peptide antibody in the antibody-enzyme (horseradish peroxidase) conjugate
solution. The standards and test specimen (serum) are added to the C-Peptide antibody coated microtiter wells. Then anti-C-Peptide antibody labeled with horseradish peroxidase (conjugate) is added. If human C-Peptide is present in the specimen, it will combine with the antibody on the well and the enzyme conjugate resulting in the C-Peptide molecules being sandwiched between the solid phase and enzymelinked antibodies. After a 1-hour incubation at room temperature, the wells are washed with water to remove unbound labeled antibodies. A solution of chemiluminescent substrate is then added and read relative light units (RLU) in a Luminometers. The intensity of the emitting light is proportional to the amount of enzyme present and is directly related to the amount of C-peptide in the sample. By reference to a series of C -peptide standards assayed in the same way, the concentration of C-peptide in the unknown sample is quantified.