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Contributed by Dr. Alexei Gratchev
I used this protocol with following DuoSets from R&D systems: human IL-1ra, human IL-6, human IL-8, human TARC, human PARC, human MCP-4.
Before you start
- Dilute your samples and standarts in the same buffer
- Wash the plates thoroughly and consistently to obtain reliable results
- Always make duplicates of your samples and standards
Solutions needed
- PBS: 137mM NaCl, 2.7nM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2-7.4, sterile filtered
- Wash buffer: 0.05% Tween in PBS
- Reagent diluent: 1% BSA in PBS, sterle filtered
- Substrate: ready-to-use TMB substrate from Biomeda
- Stop solution: 0.8 N sulfuric acid
Protocol
- Dilute coating antibody in PBS 1:180 (for full 96 well plate - mix 58µl antibody and 10.5 ml PBS)
- Distribute diluted antibody in 96 well plate (MaxiSorp from Nunc) at 100µl/well
- Incubate at 4°C overnight
- Aspirate antibody solution from the plate
- Wash once with 250 µl wash buffer
- Fill the wells with 250 µl reagent diluent, incubate for 2 h on a shaker, 100rpm at RT
- Aspirate reagent diluent from the wells, wash once with 250µl wash buffer
- Add 100 µl samples or standarts per well, incubate 2 h on a shaker at RT
- Wash the wells 3 times with 250 µl wash buffer
- Dilute the detection antibody in reagent diluent 1:180 (see above)
- Add 100 µl of diluted detection antibody to the wells
- Incubate 2 h on a shaker at RT
- Wash 4 times with 250 µl wash buffer
- Dilute streptavidine-HRP conjugate in reagent diluent 1:200 (for 96 well plate - mix 52µl conjugate and 10.5 ml reagent dilutent)
- Add 100µl of diluted conjugate to each well
- Incubate 20 min on a shaker at RT
- Wash 4 times with 250 µl wash buffer
- Add 50 µl substrate to each well
- Incubate 10-20 min at RT, control the (blue) color development
- Add 50 µl stop solution to each well. The color will turn yellow
- Dtermine the optical density of each well immediately, using a microplate reader at 450 nm, if possible make a wavelength correction at 540 or 570 nm
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