Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	31.25-2000 pg/mL
Sensitivity:	18.75 pg/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human TGF- beta2 in samples. No significant cross-reactivity or interference between Human TGF- beta2 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 TGF- beta2. Standards or samples are added to the micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human TGF- beta2 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 TGF- beta2, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of 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 TGF- beta2. You can calculate the concentration of Human TGF- beta2 in the samples by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	TGFB2: TGF-beta 2 has suppressive effects on interleukin-2 dependent T-cell growth. Homodimer; disulfide-linked. Heterodimers with TGFB1 and with TGFB3 have been found in bone. Interacts with the serine proteases, HTRA1 and HTRA3. Interacts with ASPN. Belongs to the TGF-beta family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Cell development/differentiation; Motility/polarity/chemotaxis; Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 1q41 Cellular Component: extracellular matrix; extracellular space; cell soma; axon; extracellular region; endosome Molecular Function:protein binding; protein homodimerization activity; growth factor activity; protein heterodimerization activity; beta-amyloid binding; punt binding; cytokine activity; transforming growth factor beta receptor binding; receptor signaling protein serine/threonine kinase activity; receptor binding Biological Process: heart morphogenesis; extracellular matrix organization and biogenesis; collagen fibril organization; catagen; heart development; dopamine biosynthetic process; SMAD protein nuclear translocation; protein amino acid phosphorylation; cell-cell signaling; hair follicle development; transforming growth factor beta receptor signaling pathway; somatic stem cell division; cell cycle arrest; cell growth; embryonic gut development; cartilage condensation; response to drug; platelet activation; neutrophil chemotaxis; negative regulation of immune response; neuron fate commitment; positive regulation of cell cycle; positive regulation of catagen; positive regulation of cell growth; positive regulation of phosphoinositide 3-kinase cascade; cardioblast differentiation; positive regulation of protein secretion; positive regulation of cell division; activation of protein kinase activity; neuron development; response to progesterone stimulus; positive regulation of heart contraction; cell death; axon guidance; positive regulation of immune response; wound healing; cell morphogenesis; cardiac muscle cell proliferation; positive regulation of stress-activated MAPK cascade; odontogenesis; negative regulation of cell proliferation; platelet degranulation; positive regulation of neuron apoptosis; salivary gland morphogenesis; positive regulation of cell proliferation; response to wounding; hemopoiesis; angiogenesis; positive regulation of integrin biosynthetic process; negative regulation of epithelial cell proliferation; uterine wall breakdown; intercellular junction assembly and maintenance; cell migration; regulation of transforming growth factor-beta2 production; hair follicle morphogenesis; positive regulation of cell adhesion mediated by integrin; glial cell migration; positive regulation of ossification; cell proliferation; embryonic development; eye development; generation of neurons; positive regulation of cardioblast differentiation; response to hypoxia; epithelial to mesenchymal transition; blood vessel remodeling; negative regulation of cell growth; blood coagulation Disease: Loeys-dietz Syndrome 4
NCBI Summary:	This gene encodes a member of the transforming growth factor beta (TGFB) family of cytokines, which are multifunctional peptides that regulate proliferation, differentiation, adhesion, migration, and other functions in many cell types by transducing their signal through combinations of transmembrane type I and type II receptors (TGFBR1 and TGFBR2) and their downstream effectors, the SMAD proteins. Disruption of the TGFB/SMAD pathway has been implicated in a variety of human cancers. The encoded protein is secreted and has suppressive effects of interleukin-2 dependent T-cell growth. Translocation t(1;7)(q41;p21) between this gene and HDAC9 is associated with Peters' anomaly, a congenital defect of the anterior chamber of the eye. The knockout mice lacking this gene show perinatal mortality and a wide range of developmental, including cardiac, defects. Alternatively spliced transcript variants encoding different isoforms have been identified. [provided by RefSeq, Sep 2010]
UniProt Code:	P61812
NCBI GenInfo Identifier:	48429157
NCBI Gene ID:	7042
NCBI Accession:	P61812. 1
UniProt Secondary Accession:	P61812,P08112, Q15579, Q15581, Q4VAV9, B4DKC5,
UniProt Related Accession:	P61812
Molecular Weight:	Calculated MW: 47kDa/50kDaObserved MW: 55kDa
NCBI Full Name:	Transforming growth factor beta-2
NCBI Synonym Full Names:	transforming growth factor, beta 2
NCBI Official Symbol:	TGFB2
NCBI Official Synonym Symbols:	LDS4; TGF-beta2
NCBI Protein Information:	transforming growth factor beta-2; G-TSF; cetermin; polyergin; BSC-1 cell growth inhibitor; prepro-transforming growth factor beta-2; glioblastoma-derived T-cell suppressor factor
UniProt Protein Name:	Transforming growth factor beta-2
UniProt Synonym Protein Names:	BSC-1 cell growth inhibitor; Cetermin; Glioblastoma-derived T-cell suppressor factor; G-TSF; PolyerginLatency-associated peptide; LAP
Protein Family:	Transforming growth factor
UniProt Gene Name:	TGFB2
UniProt Entry Name:	TGFB2_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
2000	2.341 2.393	2.367	2.312
1000	1.675 1.679	1.677	1.622
500	0.895 0.865	0.88	0.825
250	0.48 0.502	0.491	0.436
125	0.25 0.23	0.24	0.185
62.5	0.157 0.153	0.155	0.1
31.25	0.105 0.107	0.106	0.051
0	0.045 0.065	0.055	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human TGF- beta2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human TGF- beta2 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)	110.16	263.47	859.83	114.83	248.33	855.61
Standard deviation	6.81	11.33	32.33	5.76	12.44	26.35
C V (%)	6.18	4.30	3.76	5.02	5.01	3.08

Recovery

The recovery of Human TGF- beta2 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)	93-107	100
EDTA plasma (n=5)	93-110	100
Cell culture media (n=5)	86-96	91

Linearity

Samples were spiked with high concentrations of Human TGF- beta2 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 (%)	84-98	87-100	90-104
1:2	Average (%)	90	94	95
1:4	Range (%)	92-108	81-91	87-99
1:4	Average (%)	98	86	94
1:8	Range (%)	92-106	86-100	83-95
1:8	Average (%)	100	91	88
1:16	Range (%)	88-101	83-97	83-94
1:16	Average (%)	95	88	88

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.