Human and mouse TGF beta 1 kit HTRF®

The HTRF human and mouse TGF beta 1 kit is designed for the quantification of human or mouse TGF beta 1 release in cell supernatant.

No-wash
Validated on PBMC
Under 2h bench time
Lyophilized

The HTRF human and mouse TGF beta 1 kit is designed for the quantification of human or mouse TGF beta 1 release in cell supernatant.

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Overview

TGF beta 1 (Transforming Growth Factor Beta-1) is considered one of the major cytokines involved in the regulation of extracellular matrix (ECM) synthesis and degradation, as well as a major profibrotic factor. TGF beta 1 pre-proprotein is processed to generate a latency-associated peptide (LAP) and a mature TGFß1 peptide, which remain associated through strong non-covalent interactions. An activation step (MMP2 cleavage or acid activation) is required to release the active form of TGF beta 1 from the TGFß1-LAP complex.

Assessment of serum samples often requires enhanced sensitivity. In some cases, AlphaLISA assays may have sufficient sensitivity to enable the detection of low levels of analytes in serum or plasma. Check out the Human TGF-β1 AlphaLISA kit and the TGF-β1 AlphaLisa biotin-free kit’s analytical performances for more information or learn more about AlphaLISA. When assaying, always follow the recommended protocol and avoid highly haemolyzed samples.

Benefits

SCALABLE FROM 96 TO 1536WELLS
VALIDATED ON MOUSE KUPFFER CELLS

Activation step

The samples and cell culture supernatants contain TGF beta 1 and a TGFß1-LAP complex, which require an acid activation step followed by a neutralization in order to be detected (sample activation can follow one of the protocols described on the right). The reagents needed for this activation step are provided in the kit for maximum convenience. Standards and activated samples are dispensed directly into the detection (white, low-volume) plate for the detection by HTRF® reagents.

Assay principle

Cell supernatant, sample, or standard is dispensed directly into the assay plate for the detection by HTRF® reagents (384-well low-volume white plate or Cisbio low-volume 96-well plate in 20 µl). 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 up to a 1536 well-format by simply resizing each addition volume proportionally.

Assay data analysis

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.

To fully understand how to deal with HTRF data processing and also 4PL 1/y² fitting, please visit this page.

Cisbio also worked with Myassays.com to help you in your data analysis. By clicking on this link, you’ll be able to access a free online software to run your TGF beta 1 analysis.

Technical specifications of human and mouse TGF beta 1 kit

Sample size	16 µL
Final assay volume	20 µL
Kit components	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 hTGFß value (pg/mL)
Species	Human, mouse, bovine (use of FCS is possible up to 5% max), others expected (based on sequences similarities). No detection of chicken TGFß1.
Specificity	There is no cross-reactivity with human TGFß2 and TGFß3.

Intra and inter assay

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%

Human TGFß1 secretion in PBMC cells stimulated with Ionomycin & PMA

Human PBMCs plated at 125 kcells/well (96 wells plate) in RPMI containing 4% FCS were stimulated for 18 h with Ionomycin (1 µg/mL) and PMA ranging from 0.5 to 50 ng/mL. After transfer into polypropylene microtubes, 50 µL of supernatants were treated with the acid activation reagent (5 µL, 10 min, room temperature) then with the neutralization reagent (5 µL). 16 µL were transferred into a white detection plate. Recommended controls include complemented media alone and unstimulated cells in complemented medium to determine TGF beta in stimulation media and baseline concentrations respectively.

Mouse TGFß1 secretion from immortalized Kupffer cells

Immortalized mouse Kupffer cells (ImKC) were plated at 650 kcells/well (24 wells plate) in RPMI containing 4% FCS. After 24 h resting, the cells were stimulated for 16 h with increasing concentrations of LPS ranging from 0.05 to 5 µg/mL (final volume 500 µL/well). 100 µL of supernatants were acid activated (10 µL) for 10 min at room temperature then neutralized (10 µL). 16 µL of activated supernatants were then transferred into a white detection plate (384 low volume) to be analyzed by the Human TGF beta 1 Assay.

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