Components
1.Microplate: 96 breakable wells (12strips x 8wells) coated with anti-mouse IL-1 beta.
2.20x Wash Buffer Concentmouse: 1 Vial, 25 ml.
3.5x Assay Diluent: 1vial, 15 ml.
4.Standards: 2 vials, recombinant mouse IL-1 beta.
5.Detection Antibody: 2 vials, biotinylated anti-mouse IL-1 beta.
6.HRP-Streptavidin Concentmouse: 1vial.
7.TBM Substmouse solution: 1 Vial, 12 ml.
8.Stop Solution: 1 Vial, 8 ml of 0.2 M sulfuric acid.
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
Dilute the MIX Diluent Concentrate 10-fold with reagent grade water to produce a 1x solution. Store for up to 30 days at 2-8 °C. 4 Mouse IL-1 beta Standard: Reconstitute the Mouse IL-1 beta Standard (2 ng) with 2 mL of MIX Diluent to generate a 1 ng/mL standard stock solution.
Assay Procedure
1.Add the Standard working solution to the first two columns: Each concentration of the solution is added in duplicate, to one well each, side by side (100 μL for each well).Cover the plate with the sealer provided in the kit. Incubate for 90 min at 37 °C.
2.Remove the liquid out of each well, do not wash. Immediately add 100 μL of Biotinylated Detection Ab working solution to each well. Cover with the Plate sealer. Gently mix up. Incubate for 1 hour at 37 °C.
3.Aspirate or decant the solution from each well, add 350 μL of wash buffer to each well. Soak for 1~2 min and aspirate or decant the solution from each well and pat it dry against clean absorbent paper. Repeat this wash step 3 times.
4.Add 100 μL of HRP Conjugate working solution to each well. Incubate for 30 min at 37 °C.
5.Aspirate or decant the solution from each well, repeat the wash process for five times as conducted in step 3.
6.Add 90 μL of Substrate Reagent to each well. Cover with a new plate sealer. Incubate for about 15 min at 37 °C. Protect the plate from light.
7.Add 50 μL of Stop Solution to each well.
8.Determine the optical density (OD value) of each well at once with a micro-plate reader set to 450 nm.
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 4.1 % and 7.0% respectively.
Handling Advice
This product is for Research Use Only and is not intended for use in diagnostic procedures. Prepare all reagents (diluent buffer, wash buffer, standard, biotinylated antibody, and SP conjugate) as instructed, prior to running the assay. 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. Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed. May be stored for up to 30 days in a vacuum desiccator. Diluent (1x) may be stored for up to 30 days at 2-8°C. Store Standard at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.
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. Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed. May be stored for up to 30 days in a vacuum desiccator. Diluent (1x) may be stored for up to 30 days at 2-8°C. Store Standard at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.
Restrictions
For Research Use only
