This human insulin TRF-PINCER^TM assay kit is to be used for the measurement of insulin levels in biological buffers and culture medium, for example, to monitor the insulin production in the cultured or extract of islets of Langerhans.

Insulin is a hormone responsible for regulating the metabolism of glucose and most amino acids in human body. It is synthesized in the β-cells of the islets of Langerhans, and is released after removal of the C-peptide from proinsulin¹. Human insulin consists of a 21-residue A-chain and a 30-residue B-chain, linked by two disulfide bonds. The insulin hexamer forms in the presence of Zn²⁺ ions, which is the inactive form with long-term stability^2,3. Insulin plays important roles on human metabolism by controling cellular intake of glucose, increasing DNA replication and protein synthesis, regulating enzyme activities. Diabetes mellitus develops when the body does not produce enough (type I) or properly respond to (type II) insulin.

This assay consists two human insulin specific antibodies which recognize different epitopes on human insulin. Each antibody is modified with short complementary single-stranded DNAs which associate to form a duplex in the presence of the human insulin. TR-FRET (time-resolved fluorescence resonance energy transfer) is initiated between the two fluorophores added to the end of the two DNAs.  The amount of TR-FRET signal is associated with the human insulin concentration present in the samples⁴. This assay is simple (one step) and fast (30 min). It is highly reproducible with a CV value smaller than 3.5% and a high signal to background noise ratio. The lowest detection concentration (0.5 ng/mL) is useful for quantifying human insulin in most test samples of interest including insulin secretion from islet cells, biological buffers and insulin injection solutions. In comparison to the ELISA procedure, the PINCER^TM assay is:

Easy
Mix test sample or standard with the assay solution and incubate at ~25°C
Fast
Read fluorescent signal after 30 minutes of incubation
Sensitive
Assay measures human insulin with an LOD of 0.5 ng/mL.
Consistent
Assay eliminates the variability of coated plates and rinse steps.
Flexible
Assay is adaptable to both low- and high-throughput screening formats.

Sample Size :

Samples:

Test format:

Sensitivity:

Assay range:

Assay time:

Precision:

Recovery:

0.5 to 10 µl for buffers or culture medium. 0.2 to 1 µl for serum or plasma.

Biological buffers, culture medium, serum, or plasma.

384 well plate.

0.5 ng/ml.

0.5 ng/ml – 64 ng/ml.

30 minutes.

Inter-assay precision was CV ≤ 3.5%.

When h.insulin was spiked into culture medium, the recovery of h.insulin was 100% ± 10%.

Human Insulin TRF-PINCER^TM Assay - Standard Curves

The following human insulin standard curve was prepared using the Human Insulin TRF-PINCER^TM Assay kit. This standard curve is for the purpose of illustration only, and should not be used to calculate unknowns. Please always generate your own standard curve.

Reference:

1. Steiner DF, Oyer PE (1967). The biosynthesis of insulin and a probable precursor of insulin by a human islet cell adenoma. Proc. Natl. Acad. Sci. U.S.A. 57 (2): 473–480.
2. Chang X, Jorgensen AM, Bardrum P, Led JJ (1997). Solution structures of the R6 human insulin hexamer, Biochemistry, 36 (31): 9409–22.
3. Dunn MF, (2005) Zinc-ligand interactions modulate assembly and stability of the insulin hexamer - a review, BioMetals, 18 (4): 295–303.
4. Heyduk T, & Heyduk, E, (2005). Nucleic acid-based fluorescence sensors for detecting proteins. Anal. Chem., 77:1147-1156.
5. Heyduk, E., Moxley, MM., Salvatori, A., Corbett, JA. and Heyduk, T.Homogeneous insulin and C-peptide sensors for rapid assessment of insulin and C-peptide secretion by the islets. Diabetes. 2010 Jul 9.

h.Insulin standards or samples (in total 10 µl volume) Add 10 µl of PINCERTM Assay Solution. Incubate at room temperature for 30 minutes. Read fluorescence signal.

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RESEARCH USE:

For research use only, not for use in diagnostic procedures.

Patent pending.

For 96-well format please inquire. Click Here.

Product name	Measurements	Cat. #	Protocol	MSDS	Price	Order
Human Insulin TRF-PINCERTMAssay kit	100	IHTR1010-100			$250
Human Insulin TRF-PINCERTMAssay kit	384	IHTR1010-384			$460
Human Insulin TRF-PINCERTMAssay kit	384 x 2	IHTR1010-384 x 2			$800
Human Insulin TRF-PINCERTMAssay kit	384 x 3	IHTR1010-384 x 3			$1100
384-well round-bottom low volume non-binding surface black polystyrene microplate	1 plate	163301	--		$9.50