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HTRF Human/Mouse Fibronectin Detection Kit HTRF®

The HTRF Fibronectin 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 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 conected 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 is critically dependent on a tumor's surrounding matrix and on key adhesive and/or fibrous proteins such as fibronectin.

Precision

Sample

Mean [Fibronectin] (ng/mL)

CV

1

100

6.6%

2

750

2.0%

3

1200

7.3%


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


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Sample

Mean [Fibronectin] (ng/mL)

CV

1

9145

8%

2

40418

12%

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] (ng/mL)

[Native Fibronectin] added (ng/mL)

Proportion Standard/Native

[Fibronectin] expected (ng/mL)

[Fibronectin] measured (ng/mL)

Antigen Recovery

1

774

1234

50/50

1007

1034

103%

2

774

1234

65/35

935

946

101%

3

774

1234

35/65

1073

1029

96%

4

391

634

50/50

513

538

105%

5

391

634

65/35

476

466

98%

6

391

634

35/65

549

531

97%

7

93

148

50/50

121

120

99%

8

93

148

65/35

112

113

101%

9

93

148

35/65

129

130

101%

Three levels of recombinant protein (~770 – 390 and 90  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 4h incubation time at RT and up to overnight.

Sample

[Standard Fibronectin] (ng/mL)

[Native Fibronectin] added (ng/mL)

Proportion Standard/Native

[Fibronectin] expected (ng/mL)

[Fibronectin] measured (ng/mL)

Antigen Recovery

1

774

1234

50/50

1144

1135

99%

2

774

1234

65/35

1033

1029

100%

3

774

1234

35/65

1255

1227

98%

4

391

634

50/50

546

530

97%

5

391

634

65/35

499

479

96%

6

391

634

35/65

593

599

101%

7

93

148

50/50

140

128

91%

8

93

148

65/35

126

119

94%

9

93

148

35/65

154

138

90%

Three levels of recombinant protein (~770 – 390 and 90  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 4h incubation time at RT and up to overnight.

Dilutional linearity

Dilution Factor

C obtained (ng/ml)

C expected (ng/ml)

% Recovery

Neat

329.66

329.66

100%

2

178.29

164.83

108%

4

92.82

82.41

113%

8

45.72

41.21

111%

16

23.45

20.60

114%

Samples were from  primary human lung fibroblast cell extracts diluted in the kit diluent Lysis Buffer 3 (1X). The recovery % obtained from these experiments show 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

1664.02

1664.02

100%

2

744.06

832.01

89%

4

386.78

416.01

93%

8

207.13

208.00

100%

16

113.58

104.00

109%

32

51.37

52.00

99%

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

Cross reactivities & Interference

 Recombinant Peptide

Reactivity

Interference

Plasmatic Fibronectin EDA-

Yes

/

Fibronectin EDA+

Yes

/

Fibronectin EDB+

Yes

/

EDA fragment

No

No

EDB fragment

No

No

FCS (10%)

No

-10%

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. Fetal Calf Serum (FCS) at 10% showed no cross reactivity but can interfere slightly with detection of Fibronectin  -10%. No significant cross-reactivity was observed.

 Recombinant Peptide

Interference (5000 ng/ml)

Integrin α1β5

No

Collagen

No

Anastellin

-10%

Heparin

-15%

Interference was tested by spiking 6 increasing concentrations of peptides (up to 5000 ng/mL) in a sample containing a Fibronectin concentration of 700 ng/mL. No significant interference was observed.

Modulation of secretion of Human/Mouse Fibronectin in HepG2 cell line

Human liver hepatocellular carcinoma cells (HepG2) were plated in a culture-treated 96-well plate (100,000 cells/well) in complete culture medium and incubated overnight at 37°C - 5% CO2. Cells were then incubated for 4 hours with a Protein Transport Inhibitor (1X) or not stimulated (NS). After supernatant collection, cells were lysed with 50 µL of lysis buffer #3 (1X) for 30 minutes at RT under gentle shaking.

16 µL of lysate or diluted supernatant to remain in the linear range of the assay were transferred into a low volume white plate before adding 4 µL of the HTRF Fibronectin detection antibodies. The HTRF signal was recorded after an overnight incubation.

As expected, the Protein Transport Inhibitor blocks the release of Fibronectin in supernatant and increase Fibronectin in cell content. The pharmacological effect is less important in supernatant due to accumulation of secreted soluble Fibronectin.

Validation of HTRF Human/Mouse Fibronectin Detection kit on HepG2 cell line

Human/Mouse Fibronectin 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 Fibronectin detection antibodies. The HTRF signal was recorded after an overnight incubation. TGF-ß1 treatment results in a dose dependent increase of Fibronectin expression level, which demonstrates the differentiation of fibroblasts into myofibroblasts.

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

Human/Mouse Fibronectin 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 Fibronectin detection antibodies. The HTRF signal was recorded after an overnight incubation.

TGF-ß1 treatment results in an increase of Fibronectin expression level in cell lysate and an accumulation of higher quantity in supernatant after 48h treatment.

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

Human/Mouse Fibronectin expression in a fibroblast mouse cell line

The mouse fibroblast cell line NIH/3T3 was seeded in a T175 flask, and incubated for 3 days at 37°C. After supernatant collection, the cells were lysed with 3 mL of lysis buffer #3 (1X) for 30 minutes at RT under gentle shaking. For each sample (supernatant or cell lysate), several dilutions in the kit diluent lysis buffer  #3, were assessed to determine the most appropriate conditions to remain in the linear range of the assay. 16 µL of each sample were transferred into a low volume white microplate before the addition of 4 µL of the HTRF Fibronectin detection reagents. 

Results show a nice detection of mouse Fibronectin with high concentration in cell content and supernatant. As expected, concentration in supernatant (around 30 000 ng/ml) is greater than that cell lysate (around 10 000 ng/ml) because there is an accumulation of secretion of Fibronectin. These results show a cross reactivity with mouse model.

Validation of HTRF Human/Mouse Fibronectin Detection kit on mouse cell line

Fibronectin is a large glycoprotein of the extracellular matrix that exists in two forms: insoluble (cellular) and as a soluble plasma protein. The primary functions of fibronectin are its involvement in cell adhesion, growth, migration, and differentiation as it bridges 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.

Fibronectin is known to contribute to the wound healing process, as it will accumulate at the site of an injury to help form blood clots and aid in the development of new tissues. Fibronectin levels in serum and plasma have been identified as increasing significantly after major traumas. It is regulated by inflammatory factors and growth factors such as cytokines and TGF-b1, as well as by integrin-dependent signaling. Further, significantly altered Fibronectin levels have been observed in the disease states of certain types of cancer, fibrotic disorders, and also diabetes mellitus.

TGF-ß1 signaling pathway regulating Fibronectin production

Plate Reader Requirement

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