This protocol uses an antibody specific for mouse IL- 1 0 in the E LISA method to detect mouse IL- 1 0 , and does not detect human or viral IL-10. IL-10 from other species can be detected by using an anti-IL- 1 0 antibody against other species following the same procedure. This method is highly specific and is not sensitive to the presence of other cytokines in the assay sample. Author: J.E. Colligan et al, Translated by Xuitao Cao et al. This experiment is from the "Compendium of Immunology Laboratory Guidelines".
Operation method
IL-1 0 detection assay Move basic program Material Coated antibody: purified anti-IL-10 M A b (JES-5-2A 5 mouse IgGl anti-mouse) IL-10; Phamiingen) PBS R P M M O Complete medium Sample to be tested Closure solution: 10 % (WV) FCS, PBS preparation, sterile filtered, stored at 4°C. Wash buffer: 0.05% (O V V ) Tween 20, PBS preparation, stored at room temperature for 1 to 2 weeks. Biotinylated secondary antibody (S X C l anti-mouse IL-10; Pharmingen) Secondary antibody buffer Horseradish peroxidase (H RP)-coupled streptavidin (JacksonImmunoresearch # 016-030-084) Substrate Solution Termination solution: 0.2 mol/L sodium citrate, stored at room temperature. 1 2-channel pipette Plates: 9 6-well round-bottom polyethylene X-ray microtitre plates (Dynatech, Falcon) 9 6-well polystyrene microtiter plates ELISA plate washer (e.g. Elcawash from Elcatech) Multiwell scanning spectrophotometers (dual wavelength; e.g., VMAX, Molecular Devices) Curve analysis software (e.g. Softmax from Molecular Devices or DeltaSoft from Biometallics) 1 . Prepare 1~10/ug/m l coated antibody, PBS preparation. Immediately add 50ul of Coated Antibody to each well of the assay plate with a 12-channel pipette and incubate at 37°C for 30 min. 2. In a 96-well polystyrene plate, serially dilute the IL-IO standard as follows. Add 100M1 RPMI-IO Complete Medium to columns 2 and 3 of rows B to G. Add 100M110ng/ml of IL-IO Standard to wells G 2 and G 3, mix well, and add 100Ml of it to wells F 2 and F 3. Repeat a series of multiple dilutions up to line C. Finally, dilute to wells C 2 and C 3 and discard 100^1. B 2 and B 3 (medium only) were used as negative controls. For more product details, please visit Aladdin Scientific website.
Purified mouse IL-10 standard at known concentration