Proinsulin ELISA Kits

Proinsulin is a precursor of insulin and C-peptide. It is synthesized in pancreatic islet β-cells as a single polypeptide chain consisting of 86 amino acids, with a molecular weight of approximately 9,400 Da. After synthesis, it is transferred to secretory granules. During this process, disulfide bonds are formed, and enzymatic cleavage converts proinsulin into insulin and C-peptide. A small amount of uncleaved proinsulin remains in the secretory granules and is released into the bloodstream along with insulin and C-peptide upon granule secretion. The biological activity of proinsulin is reported to be 5–10% that of insulin. Because proinsulin contains the amino acid sequences of both insulin and C-peptide, assays designed to measure insulin or C-peptide in blood may also detect proinsulin. Therefore, when blood insulin levels are quantified using immunoassays, the result is referred to as immuno-reactive insulin (IRI), which includes proinsulin and is distinct from insulin alone. The ratio of proinsulin to insulin is considered to reflect the status of insulin biosynthesis.

In patients with type 2 diabetes, fasting and glucose-stimulated blood levels of proinsulin are higher than in healthy individuals, and the proinsulin-to-insulin ratio is also elevated. Familial hyperproinsulinemia may occur when there is a defect in the enzyme responsible for converting proinsulin to insulin, or when a proinsulin variant resistant to enzymatic cleavage is produced. In such cases, proinsulin constitutes the majority of IRI in the secretory granules. Additionally, in hyperthyroidism, excessive thyroid hormones stimulate hepatic gluconeogenesis, increasing blood glucose levels. This, in turn, promotes insulin secretion and results in the release of elevated levels of uncleaved proinsulin.

Key points for measuring Proinsulin are available in "All about Insulin Assay Kits"

The LBIS™ Mouse/Rat Proinsulin ELISA Kit is designed for the quantitative measurement of mouse and rat proinsulin. It exhibits low cross-reactivity with insulin and C-peptide, allowing specific detection of proinsulin.

Kit Performance

Assay target	Proinsulin
Calibration curve range	0.156-10 pmol/L
Analysis sample	Mouse Serum/Plasma Rat Serum/Plasma
Sample volume	10 μL
Measurement duration	Approx. 5 hours
Wavelength	Primary wavelength 450 nm Reference wavelength 620 nm

Example of Calibration Curve

Cross-reactivity with Similar Peptides

Animals	Peptides	Cross-reactivity (%)
Mouse	Proinsulin	100
	Insulin	Less than sensitivity
	C-peptide	Less than sensitivity
	Leptin	Less than sensitivity
Rat	Proinsulin	100
	Insulin	Less than sensitivity
	C-peptide	Less than sensitivity
	Leptin	Less than sensitivity
Human	Proinsulin	Cross-reactivity observed
	Insulin	Less than sensitivity
	C-peptide	Less than sensitivity

*Cross-reactivity was estimated with 1,060 pg/mL of the substance.

Example of Measurement in Mouse Serum (time course following glucose loading)

Mouse: BALB/c, Female, 8-weeks old, n=5
Glucose loading: TRELAN-G50, 0.5 mg/g weight, Oral administration
Blood sampling conditions: Serum (pre or 15/30/120 minutes after glucose loading)
Fasting: 12 hours (pre)

Example: Measurement of Proinsulin in Rat Serum

Rat	Condition	Number of Sample	Measured value (pmol/L)	SD (pmol/L)
GK, Male, 16-weeks old	-	N=8	3.13	1.47
CD(SD), Female, 12-weeks old	Ad libitum feeding	N=3	3.27	1.97

* Duplicate measurement