Components
Human IL-1 alpha Microplate: A 96 well polystyrene microplate (12 strips of 8 wells) coated with a murine monoclonal antibody against IL-1 alpha. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human IL-1 alpha Standard: Human IL-1 alpha in a buffered protein base (250 pg, lyophilized). Biotinylated Human IL-1 alpha Antibody (100x): A 100-fold biotinylated polyclonal antibody against human IL-1 alpha (60 l). EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (20 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml, 2 bottles). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80 l). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).
Material not included
Microplate reader capable of measuring absorbance at 405 nm. Pipettes (1-20 µL, 20-200 µL, and multiple channel). Deionized or distilled reagent grade water. Incubator (37 °C).
Reagent Preparation
Freshly dilute all reagents and bring all reagents to room temperature before use. EIA Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the EIA Diluent Concentrate 1:10 with reagent grade water. Store for up to 30 days at 2-8 °C.
Assay Procedure
1.Bring all reagents and samples to room temperature (18 - 25°C) before use.
2.Add 100 µl of each standard and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4°C with gentle shaking.
3.Discard the solution and wash 4 times with 1x Wash Solution.
4.Add 100 µl of 1x prepared biotinylated antibody to each well. Incubate for 1 hour at room temperature with gentle shaking.
5.Discard the solution. Repeat the wash as in step 3.
6.Add 100 µl of prepared Streptavidin solution to each well. Incubate for 45 minutes at room temperature with gentle shaking.
7.Discard the solution. Repeat the wash as in step 3.
8.Add 100 µl of TMB One-Step Substrate Reagent to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.
9.Add 50 µl of Stop Solution to each well. Read at 450 nm immediately.
Calculation of Results
Calculate the mean value of the duplicate or triplicate readings for each standard and sample.
To generate a standard curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance (OD) on the y-axis.
Determine the unknown sample concentration from the standard curve and multiply the value by the dilution factor.
Assay Precision
Intra-assay and inter-assay coefficients of variation were 4.5 % and 7.1% respectively.
Handling Advice
This product is for Research Use Only and is Not For Use In Diagnostic Procedures. Prepare all reagents (working diluent buffer, wash buffer, standard, biotinylated antibody, and SP conjugate) as instructed, prior to running the assay. 2 Prepare all samples prior to running the assay.
Storage Comment
Store SP Conjugate and Biotinylated Antibody at -20°C. Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C.
Note
Store SP Conjugate and Biotinylated Antibody at -20°C. Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C.
Restrictions
For Research Use only
