QUANTITATIVE DETERMINATION NEW PRODUCT

OF HUMAN
INSULIN RECEPTOR

Human Insulin Receptor ELISA

High sensitivity (0.173 ng/ml)
Excellent analytical characteristics
Validated for human serum and plasma (EDTA, citrate, heparin) samples
Preliminary population data

DI A BE T OL OGY
ONC OL OGY
HUMAN INSULIN RECEPTOR ELISA
Introduction
Insulin receptor (IR) is an 22-disulfide linked tetrameric
tyrosin kinase receptor located in the plasma membrane
of target cells [1]. This glycoprotein is composed of two
extracellular -subunits (731 amino acids; 135 kDa)
containing the insulin binding site and two transmembrane
-subunits (620 amino acids; 95kDa) that possess intrinsic
tyrosine kinase activity in their intracellular domains and
transduce the insulin signal into the cell interior [1,2].
The human insulin receptor is involved on glucose
homeostasis, cell growth and differentiation [3]. Binding of
insulin leads to a conformational change of the receptor,
resulting in ATP binding, autophosphorylation, and subsequent
phosphorylation of insulin receptor substrate proteins that
are linked to the action of two main signalling pathways. The
PI3-K/Akt pathway is involved in the glucose transport to the
cell, induction of proliferation or inhibition of apoptosis, while
the Ras/MAPK pathway is involved mainly in the control of cell
growth and differentiation [4].
Two insulin receptor variants are produced in mammals by
alternative splicing: IR-A lacking exon 11 and the full length
IR-B. The IR-A and IR-B isoforms show different ligand binding
affinity. IR-A is a high-affinity receptor not only for insulin
but also for IGF-II, while IR-B may be considered a specific
receptor for insulin [5]. Both insulin receptor isoforms are
coexpressed in cells, and the relative abundance of IR-A
and IR-B is regulated by development stage- and tissue-
specific factors. IR-A is predominantly expressed in fetal
and cancer cells, whereas IR-B is predominantly expressed
in well-differentiated tissues including liver, adipose tissue
and skeletal muscle [6, 7]. Dysregulation of insulin receptor
splicing, i.e., increased IR-A expression in adult life, may
play an underestimated role in cancer progression. Insulin
receptor is overexpressed in several tumors, including breast,
colon, lung, ovary, and thyroid carcinomas. Moreover, human
lymphocyte-derived malignant cells, such as the IM-9 cells,
are abundantly endowed with high-affinity insulin receptors
[7].
Circulating forms of several classes of receptor molecules
and their fragments have been identified in human plasma.
The human insulin receptor was found to be secreted into
the incubation medium by various cultured cell lines [1] and
Schaefer et al. reported that transgenic mice expressing and
secreting the soluble ectodomain of human insulin receptor
into the plasma showed chronic hyperglycemia [8]. Another
study has shown that injection of the purified His-tagged
human insulin receptor -subunit into veins of mice increased
in the concentration of blood glucose [2].
QUANTITATIVE DETERMINATION OF HUMAN
INSULIN RECEPTOR

The soluble human insulin receptor ectodomain, which

contains -subunit and a extracellular part of -subunit, has
been observed in human plasma of healthy individuals and
observed at significantly elevated levels in plasma of patients
with elevated blood glucose [9]. Furthermore, the urinary
soluble insulin receptor levels in patients with diabetes were
also significantly higher than those in healthy volunteers and
were significantly correlated with both urinary resistin and
insulin levels [10].

BioVendor Human Insulin Receptor ELISA (RD191041200R)

Intended use Test principle

The RD191041200R Human Insulin Receptor ELISA is
a sandwich enzyme immunoassay for the quantitative
measurement of human insulin receptor.
The total assay time is less than 4.5 hours
The kit measures insulin receptor in human serum
and plasma (EDTA, citrate, heparin)
Assay format is 96 wells
Standard is recombinant protein based
Components of the kit are provided ready to use,
concentrated or lyophilized
Clinical application
Diabetology
Oncology
In the BioVendor Human Insulin Receptor ELISA, Standards
and samples are incubated in microtitration wells pre-coated
with polyclonal anti-human insulin receptor antibody. After
120 minutes incubation followed by washing, biotin labelled
polyclonal anti-human insulin receptor antibody is added and
incubated with the captured insulin receptor for 60 minutes.
After another washing, streptavidin-HRP conjugate is added.
After 30 minutes incubation and the last washing step, the
remaining conjugate is allowed to react with the substrate
solution (TMB). The reaction is stopped by addition of acidic
solution and absorbance of the resulting yellow product is
measured. The absorbance is proportional to the concentration
of insulin receptor. A standard curve is constructed by plotting
absorbance values against insulin receptor concentrations
of Standards and concentrations of unknown samples are
determined using this standard curve.

3.0

2.5

2.0
HUMAN INSULIN RECEPTOR ELISA
Absorbance at 450 nm

CAT. NO.: RD191041200R

1.5
Assay format Sanwich ELISA, Biotin-labelled
antibody, 96 wells/kit 1.0
Samples Serum, Plasma (EDTA, citrate,
0.5
heparin)
Standards 0.469 to 30 ng/ml 0.0
0.1 1.0 10.0 100.0
Limit of detection 0.173 ng/ml Hu Insulin Receptor (ng/ml)
 HUMAN INSULIN RECEPTOR ELISA

Precision
Intra-assay (Within-Run) (n=8) Inter-assay (Run-to-Run) (n=5)
Sample Mean SD CV Sample Mean SD CV
(ng/ml) (ng/ml) (%) (ng/ml) (ng/ml) (%)
1 15.77 1.03 6.6 1 15.39 0.86 5.6
2 30.52 1.15 3.8 2 25.90 0.63 2.4

Spiking recovery Linearity

Serum samples were spiked with different amounts of human Serum samples were serially diluted with Dilution Buffer and
insulin receptor and assayed. assayed.
Sample Observed Expected Recovery O/E Sample Dilution Observed Expected Recovery O/E
(ng/ml) (ng/ml) (%) (ng/ml) (ng/ml) (%)
13.89 - - - 28.81 - -
22.73 23.26 97.7 2x 13.95 14.40 96.8
1 1
32.41 32.64 99.3 4x 7.46 7.20 103.5
51.67 51.39 100.6 8x 3.71 3.60 102.9
22.26 - - - 34.99 - -
31.10 31.64 98.3 2x 17.43 17.50 99.6
2 2
39.94 41.01 97.4 4x 8.77 8.75 100.3
61.24 59.76 102.5 8x 4.37 4.37 103.8

Effect of sample matrix Summary of protocol

Reconstitute Master Standard and Biotin Labeled Antibody
EDTA, citrate and heparin plasma samples were compared and prepare set of Standards
to respective serum samples from the same 10 individuals.
Results are shown below: Dilute samples (5x)
Add 100 l Standards and samples
30
Incubate at RT for 2 hours/300 rpm
Serum EDTA Plasma
Citrate Plasma Heparin Plasma
Wash plate 5 times
25 Add 100 l Biotin Labelled Antibody
Incubate at RT for 1 hour/300 rpm
Concentration of Insulin Receptor (ng/ml)

20 Wash plate 5 times

Add 100 l Streptavidin-HRP Conjugate
15 Incubate at RT for 30 min/300 rpm
Wash plate 5 times
10 Add 100 l Substrate Solution
Incubate at RT for 20 min
5
Add 100 l stop solution
Read absorbance and calculate results
0
1 2 3 4 5 6 7 8 9 10
Volunteers
 QUANTITATIVE DETERMINATION OF HUMAN
INSULIN RECEPTOR

Cross-reactivity
The antibodies used in this ELISA are specific for human insulin receptor with no detectable crossreactivity to human insulin and
IGF-I at 1000 ng/ml.

Preliminary Population Data

The following results were obtained when serum samples from 154 unselected donors (89 men + 65 women) 21 - 65 years old
were assayed with the BioVendor Human Insulin Receptor ELISA in our laboratory.

Age and Sex Dependent Distribution of Hu Insulin Receptor

Sex Age n Mean Insulin Median Insulin SD Insulin Min. Insulin Max. Insulin
(years) Receptor (ng/ml) Receptor(ng/ml) Receptor (ng/ml) Receptor (ng/ml) Receptor (ng/ml)
20-29 18 19.00 18.16 7.69 11.09 48.36
30-39 26 19.92 15.02 4.51 10.88 33.41
Male
40-49 31 17.27 16.33 4.18 12.09 32.51
50-65 14 15.81 15.44 2.34 10.94 20.42
20-29 12 16.66 16.05 4.86 12.92 22.33
30-39 25 17.94 17.73 5.37 10.28 36.98
Female
40-49 20 15.96 15.16 4.19 9.88 31.16
50-61 8 16.20 15.32 3.68 11.62 23.40

60
Men (n=89) Women (n=66)

50
Concentration of Insulin Receptor (ng/ml)

40

30

20

10

0
0 10 20 30 40 50 60 70
Age (years)

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References
1. Papa V, Russo P, Gliozzo B, Goldfine ID, Vigneri R, Pezzino V. An intact and functional soluble form of the insulin receptor is secreted by cultured cells. Endocrinology. Sep
1993;133(3):1369-1376.
2. Kanezaki Y, Matsushima R, Obata T, Nakaya Y, Matsumoto T, Ebina Y. Injection of the insulin receptor alpha subunit increases blood glucose levels in mice. Biochem Biophys Res
Commun. Sep 26 2003;309(3):572-577.
3. Giudice J, Jares-Erijman EA, Leskow FC. Insulin receptor membrane retention by a traceable chimeric mutant. Cell Commun Signal. 2013;11(1):45.
4. Taniguchi CM, Emanuelli B, Kahn CR. Critical nodes in signalling pathways: insights into insulin action. Nat Rev Mol Cell Biol. Feb 2006;7(2):85-96.
5. Malaguarnera R, Belfiore A. The insulin receptor: a new target for cancer therapy. Front Endocrinol (Lausanne).2011;2:93.
6. Sacco A, Morcavallo A, Pandini G, Vigneri R, Belfiore A. Differential signaling activation by insulin and insulin-like growth factors I and II upon binding to insulin receptor isoform
A. Endocrinology. Aug 2009;150(8):3594-3602.
7. Belfiore A, Frasca F, Pandini G, Sciacca L, Vigneri R. Insulin receptor isoforms and insulin receptor/insulin-like growth factor receptor hybrids in physiology and disease. Endocr
Rev. Oct 2009;30(6):586-623.
8. Schaefer EM, Viard V, Morin J, et al. A new transgenic mouse model of chronic hyperglycemia. Diabetes. Jan 1994;43(1):143-153.
9. Obata T, Yokota I, Kazuhiro Y, et al. Soluble insulin receptor ectodomain is elevated in the plasma of patients with diabetes. Diabetes. Aug 2007;56(8):2028-2035.
10. Umehara A, Nishioka M, Obata T, Ebina Y, Shiota H, Hashida S. A novel ultra-sensitive enzyme immunoassay for soluble human insulin receptor ectodomain and its measurement
in urine from healthy subjects and patients with diabetes mellitus. Clin Biochem. Sep 2009;42(13-14):1468-1475.

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Date of issue September 2014 about BioVendor products.