Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	0.31-20 ng/mL
Sensitivity:	0.19 ng/mL
Sample volume:	100µL
Sample type:	Tissue homogenates,cell lysates and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human INSR in samples. No significant cross-reactivity or interference between Human INSR and analogues was observed.

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human INSR. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human INSR and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human INSR, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human INSR. The concentration of Human INSR in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	Receptor tyrosine kinase which mediates the pleiotropic actions of insulin. Binding of insulin leads to phosphorylation of several intracellular substrates, including, insulin receptor substrates (IRS1, 2, 3, 4), SHC, GAB1, CBL and other signaling intermediates. Each of these phosphorylated proteins serve as docking proteins for other signaling proteins that contain Src-homology-2 domains (SH2 domain) that specifically recognize different phosphotyrosine residues, including the p85 regulatory subunit of PI3K and SHP2. Phosphorylation of IRSs proteins lead to the activation of two main signaling pathways: the PI3K-AKT/PKB pathway, which is responsible for most of the metabolic actions of insulin, and the Ras-MAPK pathway, which regulates expression of some genes and cooperates with the PI3K pathway to control cell growth and differentiation. Binding of the SH2 domains of PI3K to phosphotyrosines on IRS1 leads to the activation of PI3K and the generation of phosphatidylinositol-(3, 4, 5)-triphosphate (PIP3), a lipid second messenger, which activates several PIP3-dependent serine/threonine kinases, such as PDPK1 and subsequently AKT/PKB. The net effect of this pathway is to produce a translocation of the glucose transporter SLC2A4/GLUT4 from cytoplasmic vesicles to the cell membrane to facilitate glucose transport. Moreover, upon insulin stimulation, activated AKT/PKB is responsible for: anti-apoptotic effect of insulin by inducing phosphorylation of BAD; regulates the expression of gluconeogenic and lipogenic enzymes by controlling the activity of the winged helix or forkhead (FOX) class of transcription factors. Another pathway regulated by PI3K-AKT/PKB activation is mTORC1 signaling pathway which regulates cell growth and metabolism and integrates signals from insulin. AKT mediates insulin-stimulated protein synthesis by phosphorylating TSC2 thereby activating mTORC1 pathway. The Ras/RAF/MAP2K/MAPK pathway is mainly involved in mediating cell growth, survival and cellular differentiation of insulin. Phosphorylated IRS1 recruits GRB2/SOS complex, which triggers the activation of the Ras/RAF/MAP2K/MAPK pathway. In addition to binding insulin, the insulin receptor can bind insulin-like growth factors (IGFI and IGFII). Isoform Short has a higher affinity for IGFII binding. When present in a hybrid receptor with IGF1R, binds IGF1. PubMed:12138094 shows that hybrid receptors composed of IGF1R and INSR isoform Long are activated with a high affinity by IGF1, with low affinity by IGF2 and not significantly activated by insulin, and that hybrid receptors composed of IGF1R and INSR isoform Short are activated by IGF1, IGF2 and insulin. In contrast, PubMed:16831875 shows that hybrid receptors composed of IGF1R and INSR isoform Long and hybrid receptors composed of IGF1R and INSR isoform Short have similar binding characteristics, both bind IGF1 and have a low affinity for insulin.
NCBI Summary:	This gene encodes a member of the receptor tyrosine kinase family of proteins. The encoded preproprotein is proteolytically processed to generate alpha and beta subunits that form a heterotetrameric receptor. Binding of insulin or other ligands to this receptor activates the insulin signaling pathway, which regulates glucose uptake and release, as well as the synthesis and storage of carbohydrates, lipids and protein. Mutations in this gene underlie the inherited severe insulin resistance syndromes including type A insulin resistance syndrome, Donohue syndrome and Rabson-Mendenhall syndrome. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Oct 2015]
UniProt Code:	P06213
NCBI GenInfo Identifier:	308153655
NCBI Gene ID:	3643
NCBI Accession:	P06213. 4
UniProt Secondary Accession:	P06213,Q17RW0, Q59H98, Q9UCB7, Q9UCB8, Q9UCB9,
UniProt Related Accession:	P06213
Molecular Weight:	155,146 Da
NCBI Full Name:	Insulin receptor
NCBI Synonym Full Names:	insulin receptor
NCBI Official Symbol:	INSR
NCBI Official Synonym Symbols:	HHF5; CD220
NCBI Protein Information:	insulin receptor
UniProt Protein Name:	Insulin receptor
UniProt Synonym Protein Names:	CD_antigen: CD220
UniProt Gene Name:	INSR

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration (ng/mL)	RLU	Average	Corrected
20	33917 34047	33982	33950
10	12137 12803	12470	12438
5	5702 4798	5250	5218
2.5	2420 2628	2524	2492
1.25	1458 1308	1383	1351
0.63	871 863	867	835
0.31	595 651	623	591
0	30 34	32	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human INSR were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human INSR were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (ng/mL)	0.97	2.20	8.26	1.05	2.25	7.93
Standard deviation	0.12	0.20	0.81	0.11	0.20	0.64
C V (%)	12.37	9.09	9.81	10.48	8.89	8.07

Recovery

The recovery of Human INSR spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type	Range (%)	Average Recovery (%)
Serum (n=5)	100-117	107
EDTA plasma (n=5)	96-113	103
Cell culture media (n=5)	98-116	106

Linearity

Samples were spiked with high concentrations of Human INSR and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

		Serum (n=5)	EDTA plasma (n=5)	Cell culture media (n=5)
1:2	Range (%)	99-112	102-113	87-102
1:2	Average (%)	105	108	94
1:4	Range (%)	93-105	96-110	100-113
1:4	Average (%)	98	104	106
1:8	Range (%)	88-102	103-120	85-97
1:8	Average (%)	94	109	90
1:16	Range (%)	91-108	88-102	100-118
1:16	Average (%)	99	95	108

An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro CLIA Plate(Dismountable)	8 wells ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent A	1 vial, 5 mL	4°C (shading light)
Substrate Reagent B	1 vial, 5 mL	4°C (shading light)
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids. ) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
Determine the RLU value of each well immediately.