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- beta1 in samples. No significant cross-reactivity or interference between Human TGF- beta1 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- beta1. 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- beta1 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- beta1, 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- beta1. You can calculate the concentration of Human TGF- beta1 in the samples by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	TGFB1: Multifunctional protein that controls proliferation, differentiation and other functions in many cell types. Many cells synthesize TGFB1 and have specific receptors for it. It positively and negatively regulates many other growth factors. It plays an important role in bone remodeling as it is a potent stimulator of osteoblastic bone formation, causing chemotaxis, proliferation and differentiation in committed osteoblasts. Homodimer; disulfide-linked, or heterodimer with TGFB2. Secreted and stored as a biologically inactive form in the extracellular matrix in a 290 kDa complex (large latent TGF-beta1 complex) containing the TGFB1 homodimer, the latency-associated peptide (LAP), and the latent TGFB1 binding protein-1 (LTBP1). The complex without LTBP1 is known as the'small latent TGF-beta1 complex'. Dissociation of the TGFB1 from LAP is required for growth factor activation and biological activity. Release of the large latent TGF-beta1 complex from the extracellular matrix is carried out by the matrix metalloproteinase MMP3. May interact with THSD4; this interaction may lead to sequestration by FBN1 microfibril assembly and attenuation of TGFB signaling. Interacts with the serine proteases, HTRA1 and HTRA3: the interaction with either inhibits TGFB1-mediated signaling. The HTRA protease activity is required for this inhibition. Interacts with CD109, DPT and ASPN. Activated in vitro at pH below 3. 5 and over 12. 5. Highly expressed in bone. Abundantly expressed in articular cartilage and chondrocytes and is increased in osteoarthritis (OA). Co-localizes with ASPN in chondrocytes within OA lesions of articular cartilage. Belongs to the TGF-beta family.
UniProt Protein Details:	Protein type:Secreted; Motility/polarity/chemotaxis; Secreted, signal peptide Chromosomal Location of Human Ortholog: 19q13. 1 Cellular Component: extracellular space; proteinaceous extracellular matrix; microvillus; cell surface; cell soma; axon; Golgi lumen; cytoplasm; plasma membrane; extracellular region; nucleus Molecular Function:protein binding; enzyme binding; protein homodimerization activity; growth factor activity; protein heterodimerization activity; punt binding; cytokine activity; protein N-terminus binding; glycoprotein binding; antigen binding Biological Process: extracellular matrix organization and biogenesis; positive regulation of apoptosis; positive regulation of transcription, DNA-dependent; SMAD protein nuclear translocation; female pregnancy; positive regulation of protein amino acid dephosphorylation; activation of NF-kappaB transcription factor; regulation of protein import into nucleus; positive regulation of MAP kinase activity; connective tissue replacement during inflammatory response; regulation of transforming growth factor beta receptor signaling pathway; negative regulation of ossification; cell cycle arrest; positive regulation of isotype switching to IgA isotypes; inner ear development; regulatory T cell differentiation; positive regulation of interleukin-17 production; response to drug; positive regulation of smooth muscle cell differentiation; positive regulation of chemotaxis; active induction of host immune response by virus; positive regulation of blood vessel endothelial cell migration; regulation of sodium ion transport; negative regulation of blood vessel endothelial cell migration; negative regulation of fat cell differentiation; lymph node development; positive regulation of protein secretion; positive regulation of transcription from RNA polymerase II promoter; response to progesterone stimulus; endoderm development; positive regulation of odontogenesis; myelination; negative regulation of phagocytosis; evasion of host defenses by virus; positive regulation of cellular protein metabolic process; myeloid dendritic cell differentiation; negative regulation of transcription from RNA polymerase II promoter; phosphate metabolic process; negative regulation of cell proliferation; negative regulation of T cell proliferation; ureteric bud development; regulation of DNA binding; negative regulation of release of sequestered calcium ion into cytosol; positive regulation of cell proliferation; salivary gland morphogenesis; protein kinase B signaling cascade; protein export from nucleus; inflammatory response; aging; positive regulation of exit from mitosis; epidermal growth factor receptor signaling pathway; mitotic cell cycle checkpoint; common-partner SMAD protein phosphorylation; positive regulation of phosphoinositide 3-kinase activity; positive regulation of bone mineralization; positive regulation of peptidyl-serine phosphorylation; SMAD protein complex assembly; positive regulation of protein kinase B signaling cascade; positive regulation of protein complex assembly; positive regulation of protein import into nucleus; response to hypoxia; epithelial to mesenchymal transition; negative regulation of cell growth; negative regulation of cell-cell adhesion; negative regulation of transforming growth factor beta receptor signaling pathway; negative regulation of skeletal muscle development; mononuclear cell proliferation; protein amino acid phosphorylation; regulation of cell migration; hyaluronan catabolic process; regulation of apoptosis; response to vitamin D; negative regulation of neuroblast proliferation; positive regulation of superoxide release; receptor catabolic process; transforming growth factor beta receptor signaling pathway; germ cell migration; response to glucose stimulus; chondrocyte differentiation; T cell homeostasis; defense response to fungus, incompatible interaction; negative regulation of mitotic cell cycle; cell growth; tolerance induction to self antigen; regulation of striated muscle development; platelet activation; organ regeneration; negative regulation of DNA replication; virus-host interaction; hemopoietic progenitor cell differentiation; negative regulation of transcription, DNA-dependent; positive regulation of epithelial cell proliferation; positive regulation of collagen biosynthetic process; viral infectious cycle; response to estradiol stimulus; negative regulation of cell cycle; positive regulation of histone deacetylation; response to radiation; platelet degranulation; negative regulation of protein amino acid phosphorylation; response to wounding; lipopolysaccharide-mediated signaling pathway; adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains; negative regulation of epithelial cell proliferation; intercellular junction assembly and maintenance; regulation of binding; MAPKKK cascade; cellular calcium ion homeostasis; gut development; protein import into nucleus, translocation; ATP biosynthetic process; positive regulation of histone acetylation; positive regulation of protein amino acid phosphorylation; negative regulation of myoblast differentiation; blood coagulation; positive regulation of cell migration Disease: Camurati-engelmann Disease; Cystic Fibrosis
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. Many cells have TGFB receptors, and the protein positively and negatively regulates many other growth factors. The secreted protein is cleaved into a latency-associated peptide (LAP) and a mature TGFB1 peptide, and is found in either a latent form composed of a TGFB1 homodimer, a LAP homodimer, and a latent TGFB1-binding protein, or in an active form composed of a TGFB1 homodimer. The mature peptide may also form heterodimers with other TGFB family members. This gene is frequently upregulated in tumor cells, and mutations in this gene result in Camurati-Engelmann disease. [provided by RefSeq, Oct 2009]
UniProt Code:	P01137
NCBI GenInfo Identifier:	135674
NCBI Gene ID:	7040
NCBI Accession:	P01137. 2
UniProt Secondary Accession:	P01137,Q9UCG4, A8K792,
UniProt Related Accession:	P01137
Molecular Weight:	44,341 Da
NCBI Full Name:	Transforming growth factor beta-1
NCBI Synonym Full Names:	transforming growth factor, beta 1
NCBI Official Symbol:	TGFB1
NCBI Official Synonym Symbols:	CED; LAP; DPD1; TGFB; TGFbeta
NCBI Protein Information:	transforming growth factor beta-1; TGF-beta-1; latency-associated peptide; prepro-transforming growth factor beta-1
UniProt Protein Name:	Transforming growth factor beta-1
UniProt Synonym Protein Names:
Protein Family:	Transforming growth factor
UniProt Gene Name:	TGFB1
UniProt Entry Name:	TGFB1_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	2.378 2.384	2.381	2.323
1000	1.635 1.671	1.653	1.595
500	0.965 0.945	0.955	0.897
250	0.471 0.487	0.479	0.421
125	0.252 0.242	0.247	0.189
62.5	0.161 0.157	0.159	0.101
31.25	0.104 0.116	0.11	0.052
0	0.054 0.062	0.058	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human TGF- beta1 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- beta1 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)	106.20	233.50	976.30	109.60	234.10	999.00
Standard deviation	7.20	11.90	51.70	5.60	12.40	43.00
C V (%)	6.78	5.10	5.30	5.11	5.30	4.30

Recovery

The recovery of Human TGF- beta1 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)	94-105	100
EDTA plasma (n=5)	90-104	96
Cell culture media (n=5)	87-100	92

Linearity

Samples were spiked with high concentrations of Human TGF- beta1 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 (%)	85-100	97-111	94-111
1:2	Average (%)	91	102	101
1:4	Range (%)	92-108	83-96	90-108
1:4	Average (%)	100	89	98
1:8	Range (%)	96-108	81-96	92-107
1:8	Average (%)	102	88	98
1:16	Range (%)	98-117	79-92	95-108
1:16	Average (%)	107	86	102

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 100 µL of standard solutions into the standard wells.
Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
Add 100 µL 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 100 µL 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 350 µL 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 100 µL 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 90 µL 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.

Antibodies

Anti-TGFB1 Mouse Monoclonal Antibody (CAB0291)

Anti-TGFB1 Antibody (CAB15103)

Anti-TGFB1 Antibody (CAB16640)

Anti-TGFB1 Antibody (CAB2124)

TGFB1 Antibody (PACO03227)