I wish to perform ELISA for IL-6 and IL-10 in serum samples using Elabscience kits. Kindly give a comment on the appropriate dilutions to be used for the same to get optimum results.
Hi just read their manual you will see the procedure for the assay.You can follow the procedures here.
Human IL-6 (Interleukin 6) ELISA Kit (Elabscience)
Procedure
One hundred microlitre (100μL) of Standard, Blank, or Sample will be added per well. The blank well will be added with Reference Standard & Sample diluent. Solutions will be added to the bottom of micro ELISA plates well, avoid inside wall touching and foaming as possible. It will be mixed gently. Cover the plate with sealer we provided. It will be incubated for 90 minutes at 370C.
The liquid will be removed from of each well, don't wash. 100μL of Biotinylated Detection Ab working solution will be added to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C.
Each well will be aspirated and washed, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 350μL) (a squirt bottle, multi-channel pipette, manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, Wash Buffer will be removed by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
One hundred microlitre (100μL) of HRP Conjugate working solution will be added to each well. Cover with the Plate sealer. Incubate for 30 minutes at 37°C.
The wash process will be repeated for five times.
Ninety (90μL) of Substrate Solution will be added to each well. Cover with a new Plate sealer. Incubate for about 15 minutes at 37°C. Protect the plate from light.
Fifty (50μL) of Stop Solution will be added to each well. Then, the color turns to yellow immediately. The order to add stop solution should be the same as the substrate solution.
The optical density (OD value) of each well will be determined at once, using a micro-plate reader set to 450 nm. The micro-plate reader will be opened in advance, preheat the instrument, and set the testing parameters.
(Human IL-10 (Interleukin 10) ELISA Kit (Elabscience)
Procedure
One hundred microlitre (100μL) of Standard, Blank, or Sample will be added per well. The blank well will be added with Reference Standard & Sample diluent. Solutions will be added to the bottom of micro ELISA plates well, avoid inside wall touching and foaming as possible. It will be mixed gently. Cover the plate with sealer we provided. It will be incubated for 90 minutes at 370C.
The liquid will be removed from of each well, don't wash. 100μL of Biotinylated Detection Ab working solution will be added to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C.
Each well will be aspirated and washed, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 350μL) (a squirt bottle, multi-channel pipette, manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, Wash Buffer will be removed by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
One hundred microlitre (100μL) of HRP Conjugate working solution will be added to each well. Cover with the Plate sealer. Incubate for 30 minutes at 37°C.
The wash process will be repeated for five times.
Ninety (90μL) of Substrate Solution will be added to each well. Cover with a new Plate sealer. Incubate for about 15 minutes at 37°C. Protect the plate from light.
Fifty (50μL) of Stop Solution will be added to each well. Then, the color turns to yellow immediately. The order to add stop solution should be the same as the substrate solution.
The optical density (OD value) of each well will be determined at once, using a micro-plate reader set to 450 nm. The micro-plate reader will be opened in advance, preheat the instrument, and set the testing parameters.
Dear Sir...as recommended and what is used to be the best protocol is two fold dilution in working with serology. Since wells of microtiter plate can not take over than 250 μL approximately, hence addition of 100 μL of serum under test and 100 μL of diluting fluid provided by the kit is of best..blank is necessary...regards.
Hi, please follow manual of ELISA kit which you are using. As these data are non-parametric whatever data you will get is very crucial for statistical analysis. We also did cytokine and chemokine assays using BD diagnostic flow cytometric assays with exact protocol given by BD manual.
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