Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Mouse
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 Mouse TGF- beta1 in samples. No significant cross-reactivity or interference between Mouse 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 Mouse 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 Mouse 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 Mouse 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 Mouse TGF- beta1. You can calculate the concentration of Mouse 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:Motility/polarity/chemotaxis; Secreted; Secreted, signal peptide Cellular Component: proteinaceous extracellular matrix; extracellular space; cell surface; microvillus; cell soma; cell; axon; cytoplasm; extracellular region; nucleus; secretory granule 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; transforming growth factor beta receptor binding Biological Process: positive regulation of apoptosis; positive regulation of transcription, DNA-dependent; SMAD protein nuclear translocation; 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; regulation of transforming growth factor beta receptor signaling pathway; negative regulation of ossification; cell cycle arrest; positive regulation of isotype switching to IgA isotypes; T cell differentiation; regulatory T cell differentiation; positive regulation of interleukin-17 production; regulation of CD4-positive, CD25-positive, alpha-beta regulatory T cell differentiation; positive regulation of smooth muscle cell differentiation; positive regulation of chemotaxis; negative regulation of immune response; 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 cell division; regulation of MAPKKK cascade; 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; T cell activation; wound healing; positive regulation of cellular protein metabolic process; myeloid dendritic cell differentiation; negative regulation of transcription from RNA polymerase II promoter; phosphate metabolic process; response to organic substance; negative regulation of cell proliferation; CD4-positive, CD25-positive, alpha-beta regulatory T cell lineage commitment; negative regulation of T cell proliferation; mammary gland development; regulation of DNA binding; negative regulation of release of sequestered calcium ion into cytosol; positive regulation of cell proliferation; protein kinase B signaling cascade; protein export from nucleus; inflammatory response; 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 peptidyl-serine phosphorylation; SMAD protein complex assembly; regulation of cell proliferation; positive regulation of protein kinase B signaling cascade; cell proliferation; positive regulation of protein complex assembly; negative regulation of interleukin-17 production; positive regulation of protein import into nucleus; epithelial to mesenchymal transition; negative regulation of cell growth; negative regulation of cell-cell adhesion; negative regulation of skeletal muscle development; mononuclear cell proliferation; protein amino acid phosphorylation; hyaluronan catabolic process; regulation of apoptosis; negative regulation of neuroblast proliferation; transforming growth factor beta receptor signaling pathway; receptor catabolic process; positive regulation of superoxide release; germ cell migration; chondrocyte differentiation; defense response to fungus, incompatible interaction; T cell homeostasis; negative regulation of mitotic cell cycle; cell growth; tolerance induction to self antigen; regulation of striated muscle development; organ regeneration; skeletal muscle development; cell activation; organ morphogenesis; negative regulation of DNA replication; hemopoietic progenitor cell differentiation; negative regulation of transcription, DNA-dependent; positive regulation of epithelial cell proliferation; positive regulation of collagen biosynthetic process; defense response; response to estradiol stimulus; negative regulation of cell cycle; regulation of interleukin-23 production; positive regulation of histone deacetylation; negative regulation of protein amino acid phosphorylation; lipopolysaccharide-mediated signaling pathway; adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains; skeletal development; negative regulation of epithelial cell proliferation; intercellular junction assembly and maintenance; regulation of binding; MAPKKK cascade; morphogenesis of a branching structure; cellular calcium ion homeostasis; 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; negative regulation of T cell activation; growth; positive regulation of cell migration
UniProt Code:	P04202
NCBI GenInfo Identifier:	135675
NCBI Gene ID:	21803
NCBI Accession:	P04202. 1
UniProt Related Accession:	P04202
Molecular Weight:	44,310 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:	Tgfb; Tgfb-1; TGFbeta1; TGF-beta1
NCBI Protein Information:	transforming growth factor beta-1; TGF-beta 1; TGF-beta-1; regulatory protein; 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_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
2000	2.516 2.536	2.526	2.447
1000	1.698 1.716	1.707	1.628
500	0.952 0.928	0.94	0.861
250	0.507 0.507	0.507	0.428
125	0.273 0.261	0.267	0.188
62.5	0.186 0.178	0.182	0.103
31.25	0.123 0.141	0.132	0.053
0	0.074 0.084	0.079	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse 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 Mouse 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)	95.20	254.00	667.10	95.80	270.00	634.70
Standard deviation	5.10	11.40	28.70	5.70	11.10	31.10
C V (%)	5.36	4.49	4.30	5.95	4.11	4.90

Recovery

The recovery of Mouse 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)	93-109	101
EDTA plasma (n=5)	93-110	101
Cell culture media (n=5)	93-107	99

Linearity

Samples were spiked with high concentrations of Mouse 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 (%)	90-101	89-103	97-113
1:2	Average (%)	95	96	104
1:4	Range (%)	96-108	85-97	91-105
1:4	Average (%)	102	91	98
1:8	Range (%)	96-112	85-100	97-111
1:8	Average (%)	103	92	104
1:16	Range (%)	98-113	83-95	95-109
1:16	Average (%)	105	88	100

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.