Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	1.56-100 ng/mL
Sensitivity:	0.94 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human IL7R ELISA Kit in samples. No significant cross-reactivity or interference between Human IL7R ELISA Kit and analogues was observed.

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human IL7R. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human IL7R 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 IL7R, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human IL7R. The concentration of Human IL7R in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	IL7R: Receptor for interleukin-7. Also acts as a receptor for thymic stromal lymphopoietin (TSLP). Defects in IL7R are a cause of severe combined immunodeficiency autosomal recessive T-cell-negative/B-cell- positive/NK-cell-positive (T(-)B(+)NK(+) SCID). A form of severe combined immunodeficiency (SCID), a genetically and clinically heterogeneous group of rare congenital disorders characterized by impairment of both humoral and cell-mediated immunity, leukopenia, and low or absent antibody levels. Patients present in infancy recurrent, persistent infections by opportunistic organisms. The common characteristic of all types of SCID is absence of T-cell-mediated cellular immunity due to a defect in T-cell development. Genetic variations in IL7R are a cause of susceptibility to multiple sclerosis type 3 (MS3). A multifactorial, inflammatory, demyelinating disease of the central nervous system. Sclerotic lesions are characterized by perivascular infiltration of monocytes and lymphocytes and appear as indurated areas in pathologic specimens (sclerosis in plaques). The pathological mechanism is regarded as an autoimmune attack of the myelin sheat, mediated by both cellular and humoral immunity. Clinical manifestations include visual loss, extra-ocular movement disorders, paresthesias, loss of sensation, weakness, dysarthria, spasticity, ataxia and bladder dysfunction. Genetic and environmental factors influence susceptibility to the disease. A polymorphism at position 244 strongly influences susceptibility to multiple sclerosis. Overtransmission of the major 'C' allele coding for Thr-244 is detected in offspring affected with multiple sclerosis. In vitro analysis of transcripts from minigenes containing either 'C' allele (Thr-244) or 'T' allele (Ile-244) shows that the 'C' allele results in an approximately two-fold increase in the skipping of exon 6, leading to increased production of a soluble form of IL7R. Thus, the multiple sclerosis associated 'C' risk allele of IL7R would probably decrease membrane-bound expression of IL7R. As this risk allele is common in the general population, some additional triggers are probably required for the development and progression of MS. Belongs to the type I cytokine receptor family. Type 4 subfamily. 4 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Membrane protein, integral; Receptor, cytokine Chromosomal Location of Human Ortholog: 5p13 Cellular Component: external side of plasma membrane; extracellular region; integral to membrane; plasma membrane Molecular Function:antigen binding; interleukin-7 receptor activity; protein binding Biological Process: B cell proliferation; cell growth; cell morphogenesis; cell surface receptor linked signal transduction; homeostasis of number of cells; immune response; immunoglobulin production; lymph node development; negative regulation of T cell mediated cytotoxicity; positive regulation of T cell differentiation in the thymus; regulation of cell size; regulation of DNA recombination; signal transduction; T cell differentiation Disease: Severe Combined Immunodeficiency, Autosomal Recessive, T Cell-negative, B Cell-positive, Nk Cell-positive
NCBI Summary:	The protein encoded by this gene is a receptor for interleukin 7 (IL7). The function of this receptor requires the interleukin 2 receptor, gamma chain (IL2RG), which is a common gamma chain shared by the receptors of various cytokines, including interleukins 2, 4, 7, 9, and 15. This protein has been shown to play a critical role in V(D)J recombination during lymphocyte development. Defects in this gene may be associated with severe combined immunodeficiency (SCID). Alternatively spliced transcript variants have been found. [provided by RefSeq, Dec 2015]
UniProt Code:	P16871
NCBI GenInfo Identifier:	215274000
NCBI Gene ID:	3575
NCBI Accession:	P16871. 2
UniProt Secondary Accession:	P16871,Q05CU8, Q6NSP4, Q6NWM0, Q6NWM1, Q6NWM2, Q6NWM3 Q6SV45, Q9UPC1, B2RCS6, B4DVT1,
UniProt Related Accession:	P16871
Molecular Weight:	28,724 Da
NCBI Full Name:	Interleukin-7 receptor subunit alpha
NCBI Synonym Full Names:	interleukin 7 receptor
NCBI Official Symbol:	IL7R
NCBI Official Synonym Symbols:	ILRA; CD127; IL7RA; CDW127; IL-7R-alpha
NCBI Protein Information:	interleukin-7 receptor subunit alpha
UniProt Protein Name:	Interleukin-7 receptor subunit alpha
UniProt Synonym Protein Names:	CDw127; CD_antigen: CD127
Protein Family:	Interleukin-7 receptor
UniProt Gene Name:	IL7R
UniProt Entry Name:	IL7RA_HUMAN

As the OD 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)	O.D	Average	Corrected
100	2.29 2.32	2.305	2.248
50	1.575 1.611	1.593	1.536
25	0.892 0.884	0.888	0.831
12.5	0.429 0.443	0.436	0.379
6.25	0.252 0.248	0.25	0.193
3.13	0.158 0.146	0.152	0.095
1.56	0.098 0.114	0.106	0.049
0	0.049 0.065	0.057	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human IL7R ELISA Kit were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human IL7R ELISA Kit 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)	5.53	12.17	40.49	5.11	13.33	39.07
Standard deviation	0.38	0.65	1.60	0.30	0.69	1.96
C V (%)	6.87	5.34	3.95	5.87	5.18	5.02

Recovery

The recovery of Human IL7R ELISA Kit 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)	94-111	101
EDTA plasma (n=5)	87-98	92
Cell culture media (n=5)	93-110	101

Linearity

Samples were spiked with high concentrations of Human IL7R ELISA Kit 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 (%)	89-104	85-101	86-98
1:2	Average (%)	95	92	91
1:4	Range (%)	86-100	84-95	85-99
1:4	Average (%)	93	90	90
1:8	Range (%)	90-104	85-100	85-97
1:8	Average (%)	95	91	90
1:16	Range (%)	89-103	87-97	82-93
1:16	Average (%)	94	92	87

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 ELISA 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	1 vial, 10 mL	4°C(shading light)
Stop Solution	1 vial, 10 mL	4°C
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 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.