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| Line 17: |
Line 17: |
| * IL-8 antibody | | * IL-8 antibody |
| * 96-wells plate (MaxiSorb) | | * 96-wells plate (MaxiSorb) |
| * [[PBS]] | | * [[PBS]] 5% |
| * [http://en.wikipedia.org/wiki/Bovine_serum_albumin BSA] | | * [http://en.wikipedia.org/wiki/Bovine_serum_albumin BSA] 5% |
| * [http://www.sanquin.pl/datasheet/m1918.pdf Pelikine Compact™ Human IL-8 ELISA kit] | | * [http://www.sanquin.pl/datasheet/m1918.pdf Pelikine Compact™ Human IL-8 ELISA kit] |
| * Washing buffer | | * Washing buffer (PBS + 0.005% TWEEN 20) |
| * IL-8 antibody | | * IL-8 antibody |
| * Streptavidin conjugated to HRP | | * Streptavidin conjugated to HRP |
| Line 30: |
Line 30: |
| ** 30 mg [http://en.wikipedia.org/wiki/3,3%E2%80%99,5,5%E2%80%99-Tetramethylbenzidine TMB] | | ** 30 mg [http://en.wikipedia.org/wiki/3,3%E2%80%99,5,5%E2%80%99-Tetramethylbenzidine TMB] |
| ** 5 mL [http://en.wikipedia.org/wiki/Dimethyl_sulfoxide DMSO] | | ** 5 mL [http://en.wikipedia.org/wiki/Dimethyl_sulfoxide DMSO] |
| | |
| ===Method=== | | ===Method=== |
| <b><u>Coating of plate</u></b> | | <b><u>Coating of plate</u></b> |
Revision as of 07:41, 26 February 2010
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 ELISA
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Summary
To determine the amount of interleukin 8 (IL-8) produced by the airway smooth muscle cells (ASMC's) ELISA is performed using the PeliKine Compact™ human IL-8 ELISA kit. In this way the effect of PKA, EPAC and CSE on the inflammatory cytokine can be determined.
Materials & Methods
Materials
- Coating buffer
- IL-8 antibody
- 96-wells plate (MaxiSorb)
- PBS 5%
- BSA 5%
- Pelikine Compact™ Human IL-8 ELISA kit
- Washing buffer (PBS + 0.005% TWEEN 20)
- IL-8 antibody
- Streptavidin conjugated to HRP
- Substrate buffer
- 1.5 sodiumacetate, resuspend in 80 mL ultrapure water
- pH 5.5
- up to 100 mL with ultrapure water
- TMB stock solution (light sensitive)
Method
Coating of plate
- Mix
- 24 mL coating buffer
- 80 μL primary antibody
- Add 100 μL per well (96-wells plate)
- Incubate ON
Preparing Sandwich
- Wash coated plate 5x 200 μL PBS
- Remove PBS
- Add 200 μL BSA
- Incubate 1h @ RT and remove BSA
- Dilute samples as shown below for each 24-wells (dilution buffer (μL)/sample (μL))
24-wells (dilution buffer (μL)/sample (μL))
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1
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2
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3
|
4
|
5
|
6
|
| A
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100/100
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100/100
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100/100
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100/100
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100/100
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100/100
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| B
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150/50
|
150/50
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150/50
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175/25
|
175/25
|
175/25
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| C
|
150/50
|
150/50
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150/50
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175/25
|
175/25
|
175/25
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| D
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150/50
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150/50
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150/50
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175/25
|
175/25
|
175/25
|
- Wash of BSA with 200 μL Washing buffer 5x
- Add 100 μL of diluted sample
- Incubate ≥1h @ RT
- Wash 5x 200 μL Washing buffer
- Remove buffer
- prepare biotinylated antibody
- Add 100 μL Biotinylated antibody
- Incubate 1h @ RT
- Remove antibody
- Wash 5x 200 μL Washing buffer
- Add 100 μL streptavidin conjugated to HRP
- Incubate 25 min. @ RT
- Remove liquid
- Wash 5x 200 μL Washing buffer
- prepare TBM substrate
- 1.2 μL H2O
- 400 μL TBM stock solution
- 24 mL substrate buffer
- Add 100 μL substrate
- Incubate 30 min. @ RT
- Stop reaction, add 100 μL stopping solution
- Measure absorbance @ 540 nm
- Check duplo standard curve
- Check if values fall within standard curve (preferred)
Notes
- Of the 96-wells plate well F1 contain a Polystyrene fragment
Results
- Standard curves were accurate (R square 1>0.99)
- Negative controls showed hardly any IL-8 secretion into the medium (see figures below)
Image 180220101: The average IL-8 secretion of donor 9 corrected for cell viability. Error bars represent standard deviation. CTR: control, CSE: Cigarette smoke extract, 8-p: 8-pcpt-2'-o-me-camp, Bnz:6-Benz-cAMP.
Image 180220102: The average IL-8 secretion of donor 12 corrected for cell viability. Error bars represent standard deviation. CTR: control, CSE: Cigarette smoke extract, 8-p: 8-pcpt-2'-o-me-camp, Bnz:6-Benz-cAMP.
- Donor 9 showed twice as low IL-8 concentrations (see figures
Conclusion
- It appears that HT31P affects the cells viability negatively.
- When comparing the effect of CSE without (HT31P) and with (HT31) PKA/AKAP interaction inhibition, it seems that when AKAP/PKA association is inhibited it decreases CSE induced IL-8 release.
- PKA/AKAP interaction is required for CSE induced IL-8 secretion.
- When inducing Epac no effect is seen when comparing the HT31 situation, however slight decrease of IL-8 production is seen when HT31P control situation receives Epac stimulation.
- When Epac can compete with PKA for the present cAMP it inhibits the increased IL-8 secretion.
- Epac does not have an influence when PKA can not have its effect
- Increased PKA activity induces inhibition of IL-8 secretion even without AKAP association
- It might be that PKA is activated prior to AKAP association, removing its necessity
- PKA/AKAP induces IL-8 secretion, however an excess of active PKA with/without AKAP association lowers IL-8 secretion
- Ht31 does not prevent the activation of PKA, only its localization close to cAMP sources and PKA effectors
- PKA is the more important effector of cAMP compared to Epac
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