TGF beta 1 Kit

TGF beta 1, also known as Transforming Growth Factor beta 1, is a member of the transforming growth factor beta superfamily of cytokines. It contains more than 30 structurally related polypeptide growth factors including TGFβs (1–3).

TGF beta 1 is considered to be one of the major cytokines involved in the regulation of extracellular matrix (ECM) synthesis and degradation, as well as being a major profibrotic factor and important for wound healing. TGFβ superfamily ligands bind to a heteromeric receptor (TGFBR1/TGFBR2) which activates the SMADs signaling pathway. TGF beta 1 pre-proprotein is proteolytically processed to generate a latency-associated peptide (LAP) and a mature TGF beta 1 peptide. The cleaved LAP and TGF beta 1 remain associated through strong non-covalent interactions. Thus, TGF beta 1 is present in biological fluids as a TGF beta1-LAP complex which requires an activation step to release the active form of TGF beta 1. This activation step is usually performed using an acid activation followed by a neutralization.

Cisbio's TGF beta 1 is a homogeneous, add and read assay to measure TGF beta 1 secretion in supernatants from human or murine cells. It offers a fast and efficient alternative to ELISA.

Sample size 16 µL
Final assay volume 20 µL
Kit components Activation and neutralization reagents, lyophilized standard, frozen detection antibodies, buffers & protocol
LOD & LOQ (in Diluent) 4 pg/mL & 19 pg/mL
Range 19 – 2,000 pg/mL
Time to result ON at RT
Calibration NIBSC (89/514) value (U/mL) = 0.017 x HTRF hTGFβ value (pg/mL)
Species Human, mouse, bovine (use of FCS is possible up to 5% max), others expected (based on sequences similarities).
Specificity There is no cross-reactivity with human TGFβ2 and TGFβ3.
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Assay principle

The assay protocol, using a Cisbio 96-well low volume or a 384-well small volume white plate (20 µL final), is described below. The samples as cell culture supernatants contain TGFβ1 in a TGFβ1-LAP complex, which requires an acid activation step followed by neutralization in order to be detected (sample activation can follow one of the procols described on the right). The reagents needed for this activation step are provided with the kit for maximum convenience. Standards and activated samples are dispensed directly into the assay plate for detection by HTRF® reagents. The antibodies labeled with the HTRF donor and acceptor are pre-mixed and added in a single dispensing step, to further streamline the assay procedure. The assay can be run in 96 to 1536-well plates by simply resizing each addition volume proportionally.

 

 

TGF beta1 human assay principle

The 4 Parameter Logistic (4PL) curve is commonly recommended for fitting an ELISA standard curve. This regression enables the accurate measurement of an unknown sample across a wider range of concentrations than linear analysis, making it ideally suited to the analysis of biological systems like cytokine releases.

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Cytokines assay principle add

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Cytokines assay principle read

(1) Assay data analysis:

  • "Guidelines for data reduction" button will show you how to deal with HTRF data processing and 4PL 1/y2 fitting.
  • "MyAssays" button will direct you to a free online software able to run TGFβ1 analysis.

 

How to use HTRF cytokine assays

How to measure multiple cytokines from 1 sample

Analytical performance

Precision

Intra assay (n=24)

Sample Mean [TGFβ1] (pg/mL) CV
1 28 7%
2 167 8%
3 704 4%
  Mean CV 6%

 

Inter assay (n=4)

Sample [TGFβ1] (pg/mL) Mean (delta ratio) CV
1 32 114 3%
2 126 410 6%
3 322 1441 2%
    Mean CV 4%

 

Assay validation

It is mandatory to use an FCS supplemented culture medium for optimum biological responses and to avoid TGFβ1 sticking to culture flasks, although we recommend using not more than 5% FCS.

1. Human TGFβ1 secretion on PBMC cells stimulated with Ionomycin & PMA.

Human PBMCs plated @ 125 kcells/well (96 well plate) in RPMI containing 4% FCS were stimulated for 18h with Ionomycin (1µg/mL) and various amounts of PMA ranging from 0.5 to 50 ng/mL. Then 50 µL of supernatants were transferred into polypropylene microtubes and treated with 5 µL of acid activation reagent. After 10 min at room temperature, 5 µL of the neutralization reagent were added and after mixing and centrifugation, 16 µL were transferred into a white detection plate to be analyzed by the TGFβ1 Assay Kit.

Recommended controls include complemented media alone to determine TGFβ1 concentration in stimulation media and unstimulated cells in complemented medium to determine baseline concentration of TGFβ1.

Unstimulated PBMC supernatant displays a high background human TGFβ1 coming from activated platelets. 

TGF beta1 human assay validation graph 1

2. Mouse TGFβ1 secretion from immortalized Kupffer cells stimulated with LPS.

Immortalized mouse Kupffer cells (ImKC) were plated @ 650 kcells/well (24 well plate) in RPMI containing 4% FCS. After 24h resting, the cells were stimulated for 16h with various concentrations of LPS ranging from 0.05 to 5 µg/mL (final volume 500µL/well). 100 µL of supernatant were acid activated (acid actication reagent:10 µL) for 10 min at room temperature, then neutralized (neutralization reagent). 16µL of activated supernatants were then transferred into a white detection plate to be analyzed by the Human TGFβ1 Assay Kit.

TGF beta1 human assay validation graph 2

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
TGF beta 1 kit - 500 tests62HTGFBPEG
TGF beta 1 kit - 10,000 tests62HTGFBPEH
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Companion products

DescriptionCat. noProduct insertMSDS
TGF beta 1 kit - 3 standards62HTGFBCD3
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TGF beta 1 kit - Standard62HTGFBCDA
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