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HTRF Human Fibronectin EDA Detection Kit HTRF®

The HTRF Fibronectin EDA kit is designed for the simple and rapid quantification of the protein in cell lysates and supernatants.

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  • All inclusive kit All inclusive kit
  • No-wash No-wash
  • Highly accurate Highly accurate

The HTRF Fibronectin EDA kit is designed for the simple and rapid quantification of the protein in cell lysates and supernatants.

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Overview

Fibronectin is a large glycoprotein of the extracellular matrix whose functions are connected to cell adhesion, growth, migration, and differentiation. Fibronectin level regulation is strongly related to inflammation and fibrotic disorders, as well as to the matrix re-organization that occurs in the environment of some cancers. Tumor containment, migration, and expansion are critically dependent on a tumor's surrounding matrix and on key adhesive and/or fibrous proteins such as fibroncetin.

The EDA domain of fibronectin is an alternatively spliced version of fibronectin that is almost entirely absent from healthy tissues, and mostly expressed in active fibroblasts at injury or inflammation sites. Because of this special feature, EDA-fibronectin is a marker that is specific to fibroblast-induced fibrotic disorders, as well as being a readout of pro-fibrotic activity in a tumor matrix environment.

Precision

Sample

Mean [Fibronectin EDA] (ng/mL)

CV

1

50

4%

2

180

3%

3

750

3%

Each of the 3 samples was measured 24 times, and the %CV was calculated for each sample.

Sample

Mean [Fibronectin EDA] (ng/mL)

CV

1

226

7%

2

649

8%

Each of the samples was measured in 4 independent experiments by 4 different operators, and the % CV was calculated for each sample.

Antigen spike and recovery

Sample

[Standard Fibronectin EDA] (ng/mL)

[Native Fibronectin EDA] added (ng/mL)

Proportion (%) Standard/Native

[Fibronectin EDA] expected (ng/mL)

[Fibronectin EDA] measured (ng/mL)

Antigen Recovery

1

181

0

100/0

90.5

92

101%

2

181

221

45/55

201

212

105%

3

181

548

25/75

364.5

357

98%

4

181

926

15/85

553.5

594

107%

5

522

0

100/0

261

253

97%

6

522

221

70/30

371.5

375

101%

7

522

548

50/50

535

499

93%

8

522

926

35/65

724

737

102%

Two levels of recombinant protein (~180 and 520  ng/mL) were added to 3 dilutions of native sample from primary human lung fibroblast cell lysates and the expected concentrations were compared to those measured in order to compute antigen recoveries (acceptance criteria: 90-110%). The recovery was good after overnight incubation at RT.

Sample

[Standard Fibronectin EDA] (ng/mL)

[Native Fibronectin EDA] added (ng/mL)

Proportion (%) Standard/Native

[Fibronectin EDA] expected (ng/mL)

[Fibronectin EDA] measured (ng/mL)

Antigen Recovery

1

181

0

100/0

90.5

101

112%

2

181

194

50/50

187.5

180

96%

3

181

512

25/75

346.5

338

98%

4

181

1160

15/85

670.5

624

93%

5

522

0

100/0

261

254

97%

6

522

194

75/25

358

359

100%

7

522

512

50/50

517

520

101%

8

522

1160

30/70

841

824

98%

Two levels of recombinant protein (~180 and 520  ng/mL) were added to 3 dilutions of native sample from primary human lung fibroblast supernatants, and the expected concentrations were compared to those measured in order to compute antigen recoveries (acceptance criteria: 90-110%). The recovery was good after overnight incubation at RT.

Dilutional linearity

Dilution Factor

C obtained (ng/ml)

C expected (ng/ml)

% Recovery

Neat

769.8

769.8

100%

2

361.72

384.88

94%

4

176.54

192.44

92%

8

81.60

96.22

85%

16

46.44

48.11

97%

32

19.86

24.06

83%

Samples were from  primary human lung fibroblast cell extracts diluted in the kit diluent Lysis Buffer 3 (1X). The recovery % obtained from these experiments showed the good dilutional linearity of the assay (dilution test acceptance criteria: 80-120%).

Dilution Factor

C obtained (ng/ml)

C expected (ng/ml)

% Recovery

Neat

888.38

888.38

100%

2

423.79

444.19

95%

4

212.88

222.10

96%

8

106.89

111.05

96%

16

57.50

55.52

104%

32

31.93

27.76

115%

Samples were from  primary human lung fibroblast supernatant diluted in the kit diluent Lysis Buffer 3 (1X). The recovery % obtained from these experiments showed the good dilutional linearity of the assay (dilution test acceptance criteria: 80-120%).

Cross reactivities & Interference

 Recombinant Peptide

Reactivity

Plasmatic Fibronectin EDA-

0.23% at 5000 ng/ml 

FCS (10%)

No

Cross reactivities were determined by using standard curves for recombinant peptides in the kit diluent Lysis Buffer 3 (1X), with concentrations ranging from 0 to 1500 ng/mL or up to 10% of Fetal calf serum (FCS). No significant cross-reactivity was observed.

 Recombinant Peptide

Interference (5000 ng/ml)

Integrin α1β5

No

Collagen

No

Anastellin

No

Heparin

No

Interference was tested by spiking 6 increasing concentrations of peptides (up to 5000 ng/mL) in a sample containing a Fibronectin EDA concentration of 1000 ng/mL. No significant interference was observed.

Human Fibronectin EDA expression upon differentiation of fibroblast into myofibroblasts

Primary human lung fibroblasts (HLFs)  were plated in a culture-treated 96-well plate (12,500 cells/well) in complete culture medium and incubated at 37°C - 5% CO2. The day after, the cells were treated with increasing concentrations of TGF-ß1 for 48 hours in serum-free culture medium supplemented with 1% BSA. After supernatant collection, the cells were lysed with 50 µL of  lysis buffer #3 for 30 minutes at RT under gentle shaking.

16 µL of lysate or supernatant diluted in lysis buffer #3 to remain in the linear range of the assay were transferred into a low volume white microplate before the addition of 4 µL of the HTRF Human Fibronectin EDA detection antibodies. The HTRF signal was recorded after an overnight incubation. TGF-ß1 treatment results in a dose dependent increase of Fibronectin EDA expression level, which demonstrates the differentiation of fibroblasts into myofibroblasts.

Validation of HTRF Human Fibronectin EDA Detection kit on MRC-5 cell lysate
Validation of HTRF Human Fibronectin EDA Detection kit on MRC-5 cell supernatant

Human Fibronectin EDA expression in cancer cell line after TGFβ1 treatment

The adenocarcinomic human alveolar basal epithelial cells, A549, was plated in a culture-treated 96-well plate (50,000 cells/well) in complete culture medium and incubated at 37°C - 5% CO2. The day after, the cells were treated with 10 ng/ml of TGF-ß1 for 48 hours in serum-free culture medium supplemented with 1% BSA. The supernatant was collected, and cells were lysed with 50 µL of lysis buffer #3. 16 µL of lysate or supernatant were transferred into a low volume white microplate before the addition of 4 µL of the HTRF Human Fibronectin EDA detection antibodies. The HTRF signal was recorded after an overnight incubation.

TGF-ß1 treatment results in an increase of Fibronectin EDA expression level in lysate and supernatant, which demonstrates an upregulation of an epithelial to mesenchymal transition (EMT) marker, as expected.

Validation of HTRF Human Fibronectin EDA Detection kit on A549 cancer cell line

EDA fibronectin roles and regulation

Fibronectin (FN) is the main adhesive protein bridging the gap between cells and the extracellular matrix (ECM). It is typically secreted by fibroblasts as part of their ECM elaboration role, and is critical to morphogenesis, tissue architecture, and wound repair.

The EDA domain of fibronectin is an alternatively spliced domain that is largely absent from normal adult tissues. Its presence is characteristic of specific tissue remodeling events including injury, inflammation, wound repair, scarring, and fibrosis. Because of this, EDA fibronectin is a key and early marker of fibroblast activation when stress, growth factors, cytokines, and tissue stiffness promote their differentiation into active myofibroblasts. Along with a-SMA contractile fibers and increased collagen excretion, EDA fibronectin is a hallmark of active fibroblasts.

Unlike regular fibronectin, the EDA domain is known to amplify inflammation and fibrotic behavior in at least two ways. The EDA domain interacts with latent TGF-b binding protein-1 (LTBP-1) which in turn stores TGF-β1 in the immediate proximity of the fibroblasts drawn to the site and increases their differentiation into contractile collagen-secreting myofibroblasts. EDA fibronectin also promotes inflammatory pathways in fibroblasts, mastocytes, T-cells, and monocytes by triggering toll-like receptors TLR4.

The current understanding is that EDA-Fn splicing is regulated by the P13K/AKT pathway as a result of TGF-b and other modulators, or via regular FN signaling or mechanical tension through integrins.

TGF-ß1 signaling pathway regulating Fibronectin production

Plate Reader Requirement

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