Product Name:	Human IL-27 ELISA Kit
Product Code:	HUFI00172
Size:	96 Assays
Alias:	IL-27, IL-27 subunit alpha, IL-27-A, IL27-A
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human IL-27 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human IL-27 and the recovery rates were calculated by comparing the measured value to the expected amount of Human IL-27 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	86-100	92
EDTA plasma(n=5)	85-104	96
UFH plasma(n=5)	86-98	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human IL-27 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	86-105%	87-101%	85-104%
EDTA plasma(n=5)	85-101%	84-96%	85-100%
UFH plasma(n=5)	83-89%	80-100%	80-95%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

Q8NEV9

UniProt Protein Function:	IL27-alpha: Cytokine with pro- and anti-inflammatory properties, that can regulate T-helper cell development, suppress T-cell proliferation, stimulate cytotoxic T-cell activity, induce isotype switching in B-cells, and that has diverse effects on innate immune cells. Among its target cells are CD4 T-helper cells which can differentiate in type 1 effector cells (TH1), type 2 effector cells (TH2) and IL17 producing helper T-cells (TH17). It drives rapid clonal expansion of naive but not memory CD4 T-cells. It also strongly synergizes with IL-12 to trigger interferon- gamma/IFN-gamma production of naive CD4 T-cells, binds to the cytokine receptor WSX-1/TCCR which appears to be required but not sufficient for IL-27-mediated signal transduction. IL-27 potentiate the early phase of TH1 response and suppress TH2 and TH17 differentiation. It induces the differentiation of TH1 cells via two distinct pathways, p38 MAPK/TBX21- and ICAM1/ITGAL/ERK- dependent pathways. It also induces STAT1, STAT3, STAT4 and STAT5 phosphorylation and activates TBX21/T-Bet via STAT1 with resulting IL12RB2 up-regulation, an event crucial to TH1 cell commitment. It suppresses the expression of GATA3, the inhibitor TH1 cells development. In CD8 T-cells, it activates STATs as well as GZMB. IL-27 reveals to be a potent inhibitor of TH17 cell development and of IL-17 production. Indeed IL-27 subunit p28 alone is also able to inhibit the production of IL17 by CD4 and CD8 T-cells. While IL-27 suppressed the development of proinflammatory Th17 cells via STAT1, it inhibits the development of anti-inflammatory inducible regulatory T-cells, iTreg, independently of STAT1. IL-27 has also an effect on cytokine production, it suppresses proinflammatory cytokine production such as IL2, IL4, IL5 and IL6 and activates suppressors of cytokine signaling such as SOCS1 and SOCS3. Apart from suppression of cytokine production, IL-27 also antagonizes the effects of some cytokines such as IL6 through direct effects on T-cells. Another important role of IL-27 is its antitumor activity as well as its antiangiogenic activity with activation of production of antiangiogenic chemokines such as IP- 10/CXCL10 and MIG/CXCL9. In vein endothelial cells, it induces IRF1/interferon regulatory factor 1 and increase the expression of MHC class II transactivator/CIITA with resulting up-regulation of major histocompatibility complex class II. IL-27 also demonstrates antiviral activity with inhibitory properties on HIV-1 replivation. Belongs to the IL-6 superfamily.
UniProt Protein Details:	Protein type:Cytokine; Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 16p11 Cellular Component: extracellular space Molecular Function:interleukin-27 receptor binding; cytokine activity; receptor binding Biological Process: regulation of T-helper 1 cell differentiation; response to bacterium; regulation of T cell proliferation; regulation of defense response to virus; innate immune response; inflammatory response; positive regulation of interferon-gamma biosynthetic process
NCBI Summary:	The protein encoded by this gene is one of the subunits of a heterodimeric cytokine complex. This protein is related to interleukin 12A (IL12A). It interacts with Epstein-Barr virus induced gene 3 (EBI3), a protein similar to interleukin 12B (IL12B), and forms a complex that has been shown to drive rapid expansion of naive but not memory CD4(+) T cells. The complex is also found to synergize strongly with interleukin 12 to trigger interferon gamma (IFNG) production of naive CD4(+) T cells. The biological effects of this cytokine are mediated by the class I cytokine receptor (WSX1/TCRR). [provided by RefSeq, Jul 2008]
UniProt Code:	Q8NEV9
NCBI GenInfo Identifier:	182701368
NCBI Gene ID:	246778
NCBI Accession:	Q8NEV9.2
UniProt Secondary Accession:	Q8NEV9,Q6P676, A0N0L2,
UniProt Related Accession:	Q8NEV9
Molecular Weight:	27,493 Da
NCBI Full Name:	Interleukin-27 subunit alpha
NCBI Synonym Full Names:	interleukin 27
NCBI Official Symbol:	IL27
NCBI Official Synonym Symbols:	p28; IL30; IL-27; IL27A; IL-27A; IL27p28
NCBI Protein Information:	interleukin-27 subunit alpha; IL27-A; IL-27-A; interleukin 30; interleukin-30; IL-27 p28 subunit; IL-27 subunit alpha
UniProt Protein Name:	Interleukin-27 subunit alpha
UniProt Synonym Protein Names:	Interleukin-30; p28
Protein Family:	Interleukin
UniProt Gene Name:	IL27
UniProt Entry Name:	IL27A_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.