Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	62.50-4000 pg/mL
Sensitivity:	37.50 pg/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human IL21 ELISA Kit in samples. No significant cross-reactivity or interference between Human IL21 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 IL21. 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 IL21 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 IL21, 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 IL21. The concentration of Human IL21 in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	IL21: Cytokine with immunoregulatory activity. May promote the transition between innate and adaptive immunity. Induces the production of IgG(1) and IgG(3) in B-cells. May play a role in proliferation and maturation of natural killer (NK) cells in synergy with IL15. May regulate proliferation of mature B- and T-cells in response to activating stimuli. In synergy with IL15 and IL18 stimulates interferon gamma production in T-cells and NK cells. During T-cell mediated immune response may inhibit dendritic cells (DC) activation and maturation. Belongs to the IL-15/IL-21 family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Cytokine; Apoptosis; Secreted, signal peptide; Secreted; Cell development/differentiation Chromosomal Location of Human Ortholog: 4q26-q27 Molecular Function:hematopoietin/interferon-class (D200-domain) cytokine receptor binding; interleukin-2 receptor binding Biological Process: cell maturation; positive regulation of B cell proliferation; positive regulation of cell proliferation; positive regulation of cytokine production; positive regulation of inflammatory response; positive regulation of interleukin-17 production; positive regulation of natural killer cell mediated cytotoxicity; positive regulation of T cell proliferation; positive regulation of tissue remodeling; positive regulation of tyrosine phosphorylation of Stat1 protein; positive regulation of tyrosine phosphorylation of Stat3 protein; tyrosine phosphorylation of Stat3 protein Disease: Immunodeficiency, Common Variable, 11
NCBI Summary:	This gene encodes a member of the common-gamma chain family of cytokines with immunoregulatory activity. The encoded protein plays a role in both the innate and adaptive immune responses by inducing the differentiation, proliferation and activity of multiple target cells including macrophages, natural killer cells, B cells and cytotoxic T cells. Dysregulation of this gene plays a role in multiple immune-mediated diseases including lupus, psoriasis and chronic inflammatory diseases. Alternatively spliced transcript variants encoding multiple isoforms have been observed for this gene. [provided by RefSeq, Dec 2011]
UniProt Code:	Q9HBE4
NCBI GenInfo Identifier:	55976599
NCBI Gene ID:	59067
NCBI Accession:	Q9HBE4. 2
UniProt Secondary Accession:	Q9HBE4,A5J0L4,
UniProt Related Accession:	Q9HBE4
Molecular Weight:	16,867 Da
NCBI Full Name:	Interleukin-21
NCBI Synonym Full Names:	interleukin 21
NCBI Official Symbol:	IL21
NCBI Official Synonym Symbols:	Za11; IL-21; CVID11
NCBI Protein Information:	interleukin-21
UniProt Protein Name:	Interleukin-21
UniProt Synonym Protein Names:	Za11
Protein Family:	Interleukin
UniProt Gene Name:	IL21
UniProt Entry Name:	IL21_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 (pg/mL)	O.D	Average	Corrected
4000	2.418 2.434	2.426	2.336
2000	1.634 1.67	1.652	1.562
1000	0.879 0.869	0.874	0.784
500	0.511 0.521	0.516	0.426
250	0.279 0.269	0.274	0.184
125	0.192 0.178	0.185	0.095
62.50	0.133 0.145	0.139	0.049
0	0.089 0.091	0.09	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human IL21 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 IL21 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 (pg/mL)	197.19	568.57	1844.06	206.38	581.47	1961.30
Standard deviation	11.46	32.07	97.74	11.25	27.15	86.49
C V (%)	5.81	5.64	5.30	5.45	4.67	4.41

Recovery

The recovery of Human IL21 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)	95-109	101
EDTA plasma (n=5)	87-103	94
Cell culture media (n=5)	94-107	99

Linearity

Samples were spiked with high concentrations of Human IL21 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 (%)	93-107	90-104	91-106
1:2	Average (%)	100	96	97
1:4	Range (%)	87-103	84-98	81-97
1:4	Average (%)	95	90	88
1:8	Range (%)	91-101	83-96	84-95
1:8	Average (%)	96	89	89
1:16	Range (%)	86-101	83-97	85-98
1:16	Average (%)	93	89	90

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.