Human IL-12 (p40) ELISA Kit (HUFI00158)

Product Name:	Human IL-12 (p40) ELISA Kit
Product Code:	HUFI00158
Size:	96 Assays
Alias:	IL-12, p40, IL12B, IL-12B, CLMF2, NKSF2
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human IL-12 concentrations in serum plasma and other biological fluids.
Sensitivity:	18.75pg/ml
Range:	31.25-2000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human IL-12 and the recovery rates were calculated by comparing the measured value to the expected amount of Human IL-12 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-101	97
EDTA plasma(n=5)	88-102	95
UFH plasma(n=5)	91-101	95

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human IL-12 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)	87-102%	86-99%	93-105%
EDTA plasma(n=5)	83-98%	89-97%	82-101%
UFH plasma(n=5)	81-96%	85-96%	81-100%

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

P29460

UniProt Protein Function:	IL12B: Cytokine that can act as a growth factor for activated T and NK cells, enhance the lytic activity of NK/lymphokine- activated killer cells, and stimulate the production of IFN-gamma by resting PBMC. Defects in IL12B are a cause of mendelian susceptibility to mycobacterial disease (MSMD); also known as familial disseminated atypical mycobacterial infection. This rare condition confers predisposition to illness caused by moderately virulent mycobacterial species, such as Bacillus Calmette-Guerin (BCG) vaccine and environmental non-tuberculous mycobacteria, and by the more virulent Mycobacterium tuberculosis. Other microorganisms rarely cause severe clinical disease in individuals with susceptibility to mycobacterial infections, with the exception of Salmonella which infects less than 50% of these individuals. The pathogenic mechanism underlying MSMD is the impairment of interferon-gamma mediated immunity, whose severity determines the clinical outcome. Some patients die of overwhelming mycobacterial disease with lepromatous-like lesions in early childhood, whereas others develop, later in life, disseminated but curable infections with tuberculoid granulomas. MSMD is a genetically heterogeneous disease with autosomal recessive, autosomal dominant or X-linked inheritance. Genetic variations in IL12B are a cause of susceptibility to psoriasis type 11 (PSORS11). Psoriasis is a common, chronic inflammatory disease of the skin with multifactorial etiology. It is characterized by red, scaly plaques usually found on the scalp, elbows and knees. These lesions are caused by abnormal keratinocyte proliferation and infiltration of inflammatory cells into the dermis and epidermis. Belongs to the type I cytokine receptor family. Type 3 subfamily.
UniProt Protein Details:	Protein type:Secreted; Cytokine; Secreted, signal peptide Chromosomal Location of Human Ortholog: 5q31.1-q33.1 Cellular Component: extracellular space; membrane; interleukin-12 complex; cytoplasm Molecular Function:hematopoietin/interferon-class (D200-domain) cytokine receptor activity; identical protein binding; interleukin-12 alpha subunit binding; protein binding; protein homodimerization activity; growth factor activity; protein heterodimerization activity; interleukin-23 receptor binding; cytokine activity; interleukin-12 receptor binding Biological Process: positive regulation of granulocyte macrophage colony-stimulating factor production; positive regulation of cell adhesion; positive regulation of T-helper 1 type immune response; negative regulation of interleukin-10 production; positive regulation of T cell mediated cytotoxicity; negative regulation of smooth muscle cell proliferation; positive regulation of interleukin-12 production; positive regulation of osteoclast differentiation; positive regulation of NK T cell proliferation; positive regulation of tyrosine phosphorylation of Stat4 protein; natural killer cell activation; positive regulation of NF-kappaB import into nucleus; sensory perception of pain; defense response to Gram-negative bacterium; positive regulation of activated T cell proliferation; positive regulation of interleukin-10 production; positive regulation of natural killer cell activation; positive regulation of tyrosine phosphorylation of Stat3 protein; positive regulation of lymphocyte proliferation; natural killer cell activation during immune response; positive regulation of T cell proliferation; negative regulation of inflammatory response to antigenic stimulus; cell cycle arrest; defense response to virus; regulation of cytokine biosynthetic process; positive regulation of memory T cell differentiation; positive regulation of interleukin-17 production; positive regulation of natural killer cell proliferation; cell migration; T-helper 1 type immune response; positive regulation of NK T cell activation; T-helper cell differentiation; positive regulation of natural killer cell mediated cytotoxicity directed against tumor cell target; cytokine and chemokine mediated signaling pathway; defense response to protozoan; positive regulation of tumor necrosis factor production; regulation of tyrosine phosphorylation of Stat1 protein; response to UV-B; positive regulation of tissue remodeling; positive regulation of interferon-gamma production; negative regulation of interleukin-17 production; positive regulation of mononuclear cell proliferation; positive regulation of tyrosine phosphorylation of Stat5 protein; sexual reproduction; interferon-gamma biosynthetic process; positive regulation of defense response to virus by host; positive regulation of inflammatory response; positive regulation of interferon-gamma biosynthetic process Disease: Immunodeficiency 29
NCBI Summary:	This gene encodes a subunit of interleukin 12, a cytokine that acts on T and natural killer cells, and has a broad array of biological activities. Interleukin 12 is a disulfide-linked heterodimer composed of the 40 kD cytokine receptor like subunit encoded by this gene, and a 35 kD subunit encoded by IL12A. This cytokine is expressed by activated macrophages that serve as an essential inducer of Th1 cells development. This cytokine has been found to be important for sustaining a sufficient number of memory/effector Th1 cells to mediate long-term protection to an intracellular pathogen. Overexpression of this gene was observed in the central nervous system of patients with multiple sclerosis (MS), suggesting a role of this cytokine in the pathogenesis of the disease. The promoter polymorphism of this gene has been reported to be associated with the severity of atopic and non-atopic asthma in children. [provided by RefSeq, Jul 2008]
UniProt Code:	P29460
NCBI GenInfo Identifier:	266320
NCBI Gene ID:	3593
NCBI Accession:	P29460.1
UniProt Related Accession:	P29460
Molecular Weight:	37,169 Da
NCBI Full Name:	Interleukin-12 subunit beta
NCBI Synonym Full Names:	interleukin 12B
NCBI Official Symbol:	IL12B
NCBI Official Synonym Symbols:	CLMF; NKSF; CLMF2; IMD28; IMD29; NKSF2; IL-12B
NCBI Protein Information:	interleukin-12 subunit beta; CLMF p40; IL-12 subunit p40; IL12, subunit p40; interleukin 12, p40; interleukin-12 beta chain; NK cell stimulatory factor chain 2; cytotoxic lymphocyte maturation factor 40 kDa subunit; natural killer cell stimulatory factor, 40 kD subunit; interleukin 12B (natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2, p40)
UniProt Protein Name:	Interleukin-12 subunit beta
UniProt Synonym Protein Names:	Cytotoxic lymphocyte maturation factor 40 kDa subunit; CLMF p40; IL-12 subunit p40; NK cell stimulatory factor chain 2; NKSF2
UniProt Gene Name:	IL12B
UniProt Entry Name:	IL12B_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.