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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
- BSA
- Pelikine Compact™ Human IL-8 ELISA kit
- Washing buffer
- 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
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4
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5
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6
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| 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
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150/50
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150/50
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175/25
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175/25
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175/25
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| C
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150/50
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150/50
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150/50
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175/25
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175/25
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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
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175/25
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175/25
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- 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
Conclusion
Related
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ELISA
