Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Mouse
Detection Method:	Colormetric
Detection Range:	15.63-1000 pg/mL
Sensitivity:	9.38 pg/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Mouse IL12/p40 ELISA Kit in samples. No significant cross-reactivity or interference between Mouse IL12/p40 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 Mouse IL12/p40. 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 Mouse IL12/p40 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 Mouse IL12/p40, 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 Mouse IL12/p40. The concentration of Mouse IL12/p40 in samples can be calculated by comparing the OD of the samples to the standard curve.

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, signal peptide; Cytokine; Secreted Cellular Component: cytoplasm; extracellular space; interleukin-12 complex Molecular Function:cytokine activity; growth factor activity; hematopoietin/interferon-class (D200-domain) cytokine receptor binding; identical protein binding; interleukin-12 alpha subunit binding; interleukin-12 receptor binding; interleukin-23 receptor binding; protein binding; protein heterodimerization activity; protein homodimerization activity Biological Process: cell cycle arrest; cell migration; cell surface receptor linked signal transduction; defense response to Gram-negative bacterium; defense response to protozoan; defense response to virus; natural killer cell activation; natural killer cell activation during immune response; negative regulation of inflammatory response to antigenic stimulus; negative regulation of interleukin-10 production; negative regulation of interleukin-17 production; negative regulation of smooth muscle cell proliferation; positive regulation of activated T cell proliferation; positive regulation of cell adhesion; positive regulation of defense response to virus by host; positive regulation of granulocyte macrophage colony-stimulating factor production; positive regulation of interferon-gamma biosynthetic process; positive regulation of interferon-gamma production; positive regulation of interleukin-10 production; positive regulation of interleukin-12 production; positive regulation of interleukin-17 production; positive regulation of lymphocyte proliferation; positive regulation of mononuclear cell proliferation; positive regulation of natural killer cell activation; positive regulation of natural killer cell mediated cytotoxicity directed against tumor cell target; positive regulation of natural killer cell proliferation; positive regulation of NK T cell activation; positive regulation of NK T cell proliferation; positive regulation of osteoclast differentiation; positive regulation of T cell mediated cytotoxicity; positive regulation of T cell proliferation; positive regulation of T-helper 1 type immune response; positive regulation of tumor necrosis factor production; positive regulation of tyrosine phosphorylation of Stat3 protein; positive regulation of tyrosine phosphorylation of Stat4 protein; positive regulation of tyrosine phosphorylation of Stat5 protein; regulation of tyrosine phosphorylation of Stat1 protein; response to organic substance; response to UV-B; sensory perception of pain; T-helper cell differentiation
NCBI Summary:	This gene encodes the beta subunit p40 of the Interleukin 12 (IL-12) family of cytokines. Members of the IL-12 family form heterodimers consisting of heavy and light subunits linked by disulfide bonds. The product of this gene, p40, is a subunit of interleukins IL-12 and IL-23. [provided by RefSeq, Dec 2014]
UniProt Code:	P43432
NCBI GenInfo Identifier:	1170461
NCBI Gene ID:	16160
NCBI Accession:	P43432. 1
UniProt Secondary Accession:	P43432,Q9QUM1,
UniProt Related Accession:	P43432
Molecular Weight:	38,235 Da
NCBI Full Name:	Interleukin-12 subunit beta
NCBI Synonym Full Names:	interleukin 12b
NCBI Official Symbol:	Il12b
NCBI Official Synonym Symbols:	p40; Il-12b; Il12p40; Il-12p40
NCBI Protein Information:	interleukin-12 subunit beta
UniProt Protein Name:	Interleukin-12 subunit beta
UniProt Synonym Protein Names:	Cytotoxic lymphocyte maturation factor 40 kDa subunit; CLMF p40; IL-12 subunit p40
Protein Family:	Interleukin
UniProt Gene Name:	Il12b
UniProt Entry Name:	IL12B_MOUSE

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
1000	2.331 2.385	2.358	2.279
500	1.619 1.631	1.625	1.546
250	0.901 0.899	0.9	0.821
125	0.454 0.456	0.455	0.376
62.5	0.252 0.238	0.245	0.166
31.25	0.181 0.177	0.179	0.1
15.63	0.128 0.132	0.13	0.051
0	0.07 0.088	0.079	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse IL12/p40 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 Mouse IL12/p40 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)	50.68	116.70	379.42	52.44	116.80	384.10
Standard deviation	2.83	6.02	19.69	2.93	6.03	14.10
C V (%)	5.58	5.16	5.19	5.59	5.16	3.67

Recovery

The recovery of Mouse IL12/p40 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)	89-102	94
EDTA plasma (n=5)	84-96	90
Cell culture media (n=5)	92-107	99

Linearity

Samples were spiked with high concentrations of Mouse IL12/p40 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 (%)	93-106	87-99	92-106
1:2	Average (%)	100	94	97
1:4	Range (%)	89-100	81-95	89-102
1:4	Average (%)	94	88	94
1:8	Range (%)	86-100	85-96	86-99
1:8	Average (%)	92	90	92
1:16	Range (%)	93-105	83-96	85-97
1:16	Average (%)	99	89	92

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.