Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	15.63-1000 pg/mL
Sensitivity:	9.38 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human IDE in samples. No significant cross-reactivity or interference between Human IDE 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 IDE. 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 IDE 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 IDE, 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 IDE. The concentration of Human IDE in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	IDE: Plays a role in the cellular breakdown of insulin, IAPP, glucagon, bradykinin, kallidin and other peptides, and thereby plays a role in intercellular peptide signaling. Degrades amyloid formed by APP and IAPP. May play a role in the degradation and clearance of naturally secreted amyloid beta-protein by neurons and microglia. Belongs to the peptidase M16 family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Cell surface; EC 3. 4. 24. 56; Nuclear receptor co-regulator; Protease Chromosomal Location of Human Ortholog: 10q23. 33 Cellular Component: cell surface; cytoplasm; cytosol; extracellular space; mitochondrion; nucleus; peroxisome Molecular Function:ATP binding; glycoprotein binding; insulin binding; metalloendopeptidase activity; peptide binding; protein binding; protein homodimerization activity; receptor binding; ubiquitin binding; zinc ion binding Biological Process: beta-amyloid metabolic process; determination of adult life span; positive regulation of protein oligomerization; protein homooligomerization; proteolysis; proteolysis involved in cellular protein catabolic process
NCBI Summary:	This gene encodes a zinc metallopeptidase that degrades intracellular insulin, and thereby terminates insulins activity, as well as participating in intercellular peptide signalling by degrading diverse peptides such as glucagon, amylin, bradykinin, and kallidin. The preferential affinity of this enzyme for insulin results in insulin-mediated inhibition of the degradation of other peptides such as beta-amyloid. Deficiencies in this protein's function are associated with Alzheimer's disease and type 2 diabetes mellitus but mutations in this gene have not been shown to be causitive for these diseases. This protein localizes primarily to the cytoplasm but in some cell types localizes to the extracellular space, cell membrane, peroxisome, and mitochondrion. Alternative splicing results in multiple transcript variants encoding distinct isoforms. Additional transcript variants have been described but have not been experimentally verified. [provided by RefSeq, Sep 2009]
UniProt Code:	P14735
NCBI GenInfo Identifier:	215274252
NCBI Gene ID:	3416
NCBI Accession:	P14735. 4
UniProt Secondary Accession:	P14735,Q5T5N2, B2R721, B7ZAU2, D3DR35,
UniProt Related Accession:	P14735
Molecular Weight:	118kDa
NCBI Full Name:	Insulin-degrading enzyme
NCBI Synonym Full Names:	insulin degrading enzyme
NCBI Official Symbol:	IDE
NCBI Official Synonym Symbols:	INSULYSIN
NCBI Protein Information:	insulin-degrading enzyme
UniProt Protein Name:	Insulin-degrading enzyme
UniProt Synonym Protein Names:	Abeta-degrading protease; Insulin protease; Insulinase; Insulysin
Protein Family:	IgM protease
UniProt Gene Name:	IDE

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 (pg/mL)	RLU	Average	Corrected
1000	51347 56003	53675	53650
500	21304 23674	22489	22464
250	10539 9883	10211	10186
125	4615 5187	4901	4876
62.5	2470 2436	2453	2428
31.25	1331 1231	1281	1256
15.63	686 730	708	683
0	25 25	25	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human IDE 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 (pg/mL)	49.47	111.52	391.00	48.69	108.49	395.33
Standard deviation	6.33	9.32	25.42	4.27	10.66	43.37
C V (%)	12.80	8.36	6.50	8.77	9.83	10.97

Recovery

The recovery of Human IDE 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)	86-99	92
EDTA plasma (n=5)	98-113	104
Cell culture media (n=5)	88-101	94

Linearity

Samples were spiked with high concentrations of Human IDE 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 (%)	91-105	94-108	86-97
1:2	Average (%)	98	99	91
1:4	Range (%)	101-116	87-101	103-117
1:4	Average (%)	107	93	109
1:8	Range (%)	87-101	98-111	91-103
1:8	Average (%)	92	104	96
1:16	Range (%)	88-99	96-113	103-119
1:16	Average (%)	94	103	109

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.